Enzymatic Transamination of Cyclopamine Analogs

ABSTRACT

Provided are processes for the synthesis of amino analogues from ketone starting materials.

This application is a continuation of U.S. application Ser. No.13/175,053, filed Jul. 1, 2011, which is a continuation of InternationalApplication No. PCT/US2010/044597, filed Aug. 5, 2010, which claims thebenefit of U.S. Provisional Application No. 61/231,439, filed on Aug. 5,2009, each of these prior applications is incorporated herein byreference in its entirety.

BACKGROUND

Cyclopamine, a natural product isolated from Veratrum californicum, hasemerged as a significant pharmacological tool to validate the Hedgehog(Hh) pathway in cancer. Cyclopamine directly acts on SMO and inhibitstumor growth in several murine models of pancreatic, medulloblastoma,prostate, small cell lung, and digestive tract cancers. However, theclinical development of cyclopamine as a therapeutic in cancer ishampered by its poor solubility, acid sensitivity, and weak potencyrelative to other reported small-molecule Hh antagonists.

There has been considerable focus on the development of novelcyclopamine analogues with improved potency, and improvedpharmacokinetic and pharmaceutical properties relative to cyclopamine(see, for example, U.S. Pat. Nos. 7,230,004 and 7,407,967, incorporatedherein by reference). From that effort, a seven-membered D-ringsulfonamide analogue of cyclopamine, IPI-926, emerged as a clinicaldevelopment candidate (see Tremblay et al., “Discovery of a Potent andOrally Active Hedgehog Pathway Antagonist (IPI-926)” J. Med. Chem.(2009) 52:4400-4418, incorporated herein by reference). Large quantitiesof IPI-926 are required for clinical development. Moreover, otherpromising amino analogues can be synthesized following routes similar tothat used to generate IPI-926.

SUMMARY

Provided are novel processes for the synthesis of amino analogues, suchas IPI-926, from ketone starting materials.

For example, in one aspect, provided is a process for preparing acompound of formula (II):

-   -   or a salt thereof;    -   from a compound of formula (I):

-   -   or a salt thereof;    -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,        R¹⁴, R¹⁵ and X are as defined herein,    -   the process comprising contacting a compound of formula (I) or a        salt thereof, an amino donor molecule, and an amine transaminase        enzyme in a solution to provide a compound of formula (II) or a        salt thereof.

In certain embodiments, the process further comprises adding a co-factorto the solution.

In certain embodiments, the co-factor is pyridoxal phosphate (PLP).

In certain embodiments, the co-factor is a coenzyme. In certainembodiments, the co-enzyme is selected from Lalanine dehydrogenase(LADH), lactate dehydrogenase (LDH), nicotinamide adenine dinucleotidephosphate (NADPH), nicotinamide adenine dinucleotide (NAD), formatedehydrogenase (FDH), and glucose dehydrogenase (GDH).

In certain embodiments, process further comprises adding a pyruvatereductase mix to the solution. As used herein, the term “pyruvatereductase mix” refers to a combination that includes an enzyme that iscapable of mediating the reduction of pyruvate and one or more (e.g., 1,2, 3, 4, 5, or 6, e.g., 2, 3 or 4, e.g., 2 or 3) additional agents(e.g., an enzyme, a co-enzyme or co-factor, a reducing agent as well ascombinations thereof).

In embodiments, the enzyme that is capable of mediating the reduction ofpyruvate is LDH.

In embodiments, the combination includes an agent (e.g., an enzyme) thatis capable of regenerating a co-enzyme or co-factor that mediatespyruvate reduction. In certain embodiments, the combination can includean enzyme that is capable of regenerating NADPH. Such enzymes caninclude, without limitation, GDH and FDH.

In embodiments, the combination includes a reducing agent. Reducingagents can include, without limitation, glucose or formate.

In embodiments, the combination includes a co-enzyme or co-factor.Co-enzymes or co-factors can include, without limitation, NAD.

In embodiments, the combination includes an agent (e.g., an enzyme) thatis capable of regenerating a co-enzyme or co-factor that mediatespyruvate reduction as defined anywhere herein; and one or moreadditional agents (e.g., 2 or 3 additional agents). For example, thecombination can include an agent (e.g., an enzyme) that is capable ofregenerating a co-enzyme or co-factor that mediates pyruvate reductionas defined anywhere herein; and one or both of the following: a reducingagent and a co-enzyme or co-factor.

In embodiments, a pyruvate reductase mix includes LDH, an agent (e.g.,an enzyme) that is capable of regenerating a co-enzyme or co-factor thatmediates pyruvate reduction as defined anywhere herein; and one or moreadditional agents (e.g., 2 or 3 additional agents); e.g., one or both ofthe following: a reducing agent and a co-enzyme or co-factor. Forexample, a pyruvate reductase mix can include LDH, GDH, and glucose andcan further include NAD, e.g., PRM-102 (Codexis), which includes LDH,GDH, glucose, and NAD⁺. As another example, a pyruvate reductase mix caninclude LDH, FDH, and formate and can further include NAD.

In certain embodiments, when pyruvate is generated during the course ofthe processes described herein, the pyruvate can be removed, chemicallyconverted to another product and optionally further removed; or recycled(or combinations thereof). Methods describing such operations aredescribed in, e.g., Koszelewski, D., et al., Trends in Biotechnology2010, 28, 324-332, which is incorporated herein by reference in itsentirety.

In certain embodiments, the enzyme preferentially generates a compoundof formula (II), or a salt thereof, wherein the newly-formed amino grouphas (R) or (S) stereochemistry.

In certain embodiments, the enzyme is an omega amine transaminase, abroad-range transaminase, a glutamate-pyruvate transaminase or aglutamate-oxaloacetic transaminase.

In certain embodiments, the enzyme is an omega amine transaminase.

In certain embodiments, the omega amine transaminase is selected fromthe group consisting of ATA-101, ATA-102, ATA-103, ATA-104, ATA-105,ATA-106, ATA-107, ATA-108, ATA-109, ATA-110, ATA-113, ATA-114, ATA-115,ATA-116, ATA-117, ATA-124, an omega amine transaminase fromChromobacterium violaceum, an omega amine transaminase from Alcaligenesdenitrificans, an omega amine transaminase from Arthrobactercitreus, anomega amine transaminase from Klebsiella pneumoniae, an omega aminetransaminase from Bacillus thuringiensis, an omega amine transaminasefrom Bacillus cereus, and an omega amine transaminase from Vibriofluvialis.

In certain embodiments, the amino donor molecule is an amine or a saltthereof. In certain embodiments, the amine is selected frompyridoxamine, methylbenzylamine, 2-aminobutane, propyl amine, isopropylamine, 1,1,1-trifluoropropan-2-amine,1,1,1,3,3,3-hexafluoropropan-2-amine, benzyl amine, 2-amino-1-butanol,1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane,1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane,1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane,dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-1-(2-naphthyl)ethane,cis-2-methylcyclopentanamine, trans-2-methylcyclopentanamine,cis-3-methylcyclopentanamine, trans-3-methylcyclopentanamine,cis-2-ethylcyclopentanamine, trans-2-ethylcyclopentanamine,cis-3-ethylcyclopentanamine, trans-3-ethylcyclopentanamine,cis-2-methylcyclohexanamine, trans-2-methylcyclohexanamine,cis-3-methylcyclohexanamine, trans-3-methylcyclohexanamine,cis-2-ethylcyclohexanamine, trans-2-ethylcyclohexanamine,cis-3-ethylcyclohexanamine, trans-3-ethylcyclohexanamine,1-aminotetralin, 2-aminotetralin, 2-amino-5-methoxytetralin,1-aminoindane, 2-aminoindane, 2-amino-1-propanol, cis-1-amino-2-indanol,trans-1-amino-2-indanol, 1-amino-6-hydroxyindanamine, taurine, or a saltthereof.

In certain embodiments, the amino donor molecule is a chiral amino donormolecule. In certain embodiments, the chiral amino donor molecule is achiral amine or salt thereof, e.g., an amine containing at least oneasymmetric center. Exemplary chiral amines include, but are not limitedto, (R)-methylbenzylamine, (S)-methylbenzylamine, (S)-2-aminobutane,(R)-2-aminobutane, (S)-1-aminoindane, (R)-1-aminoindane,(R)-1,1,1-trifluoropropan-2-amine, (S)-1,1,1-trifluoropropan-2-amine,(R)-2-amino-1-propanol, (S)-2-amino-1-propanol,(1R,2S)-cis-1-amino-2-indanol, (1R,2R)-trans-1-amino-2-indanol,1-(R)-amino-6-hydroxyindanamine, 1-(S)-amino-6-hydroxyindanamine,(R)-2-amino-1-butanol, (S)-2-amino-1-butanol,(R)-1-amino-1-phenylethane, (S)-1-amino-1-phenylethane,(R)-1-amino-1-(2-methoxy-5-fluorophenyl)ethane,(S)-1-amino-1-(2-methoxy-5-fluorophenyl)ethane,(R)-1-amino-1-phenylpropane, (S)-1-amino-1-phenylpropane,(R)-1-amino-1-(4-hydroxyphenyl)-propane,(S)-1-amino-1-(4-hydroxyphenyl)-propane,(R)-1-amino-1-(4-bromophenyl)propane,(S)-1-amino-1-(4-bromophenyl)propane,(R)-1-amino-1-(4-nitrophenyl)propane,(S)-1-amino-1-(4-nitrophenyl)propane, (R)-1-phenyl-2-aminopropane,(S)-1-phenyl-2-aminopropane,(R)-1-(3-trifluoromethylphenyl)-2-aminopropane,(S)-1-(3-trifluoromethylphenyl)-2-aminopropane(R)-1-amino-1-phenylbutane, (S)-1-amino-1-phenylbutane,(R)-1-phenyl-2-aminobutane, (S)-1-phenyl-2-aminobutane,(R)-1-(2,5-di-methoxy-4-methylphenyl)-2-aminobutane,(S)-1-(2,5-di-methoxy-4-methylphenyl)-2-aminobutane,(R)-1-phenyl-3-aminobutane, (S)-1-phenyl-3-aminobutane,(R)-1-(4-hydroxyphenyl)-3-aminobutane,(S)-1-(4-hydroxyphenyl)-3-aminobutane, (R)-1-amino-1-(2-naphthyl)ethane,(S)-1-amino-1-(2-naphthyl)ethane (R)-1-aminotetralin,(S)-1-aminotetralin, (R)-2-aminotetralin, (S)-2-aminotetralin,(R)-2-amino-5-methoxytetralin, (S)-2-amino-5-methoxytetralin,(1R,2S)-cis-2-methylcyclopentanamine,(1S,2R)-cis-2-methylcyclopentanamine,(1R,2R)-trans-2-methylcyclopentanamine,(1S,2S)-trans-2-methylcyclopentanamine,(1R,3S)-cis-3-methylcyclopentanamine,(1S,3R)-cis-3-methylcyclopentanamine,(1R,3R)-trans-3-methylcyclopentanamine,(1S,3S)-trans-3-methylcyclopentanamine,(1R,2S)-cis-2-ethylcyclopentanamine,(1S,2R)-cis-2-ethylcyclopentanamine,(1R,2R)-trans-2-ethylcyclopentanamine,(1S,2S)-trans-2-ethylcyclopentanamine,(1R,3S)-cis-3-ethylcyclopentanamine,(1S,3R)-cis-3-ethylcyclopentanamine,(1R,3R)-trans-3-ethylcyclopentanamine,(1S,3S)-trans-3-ethylcyclopentanamine,(1R,2S)-cis-2-methylcyclohexanamine,(1S,2R)-cis-2-methylcyclohexanamine,(1R,2R)-trans-2-methylcyclohexanamine,(1S,2S)-trans-2-methylcyclohexanamine,(1R,3S)-cis-3-methylcyclohexanamine,(1S,3R)-cis-3-methylcyclohexanamine,(1R,3R)-trans-3-methylcyclohexanamine,(1S,3S)-trans-3-methylcyclohexanamine,(1R,2S)-cis-2-ethylcyclohexanamine, (1S,2R)-cis-2-ethylcyclohexanamine,(1R,2R)-trans-2-ethylcyclohexanamine,(1S,2S)-trans-2-ethylcyclohexanamine,(1R,3S)-cis-3-ethylcyclohexanamine, (1S,3R)-cis-3-ethylcyclohexanamine,(1R,3R)-trans-3-ethylcyclohexanamine,(1S,3S)-trans-3-ethylcyclohexanamine, or a salt thereof.

In certain embodiments, the amino donor molecule is an amino acid or apolypeptide thereof and/or salt thereof. In certain embodiments, theamino acid is selected from glycine, alanine, aspartic acid,phenylalanine, 2-aminopentanedioic acid, 3-aminobutyrate,γ-aminobutyrate, β-alanine, asparagine, cysteine, glutamic acid,glutamine, proline, selenocysteine, serine, tyrosine, arginine,histidine, ornithine, isoleucine, leucine, lysine, methionine,threonine, tryptophan, valine, and polypeptides thereof and/or saltsthereof.

In certain embodiments, the chiral amino donor molecule is a chiralamino acid or a polypeptide thereof and/or a salt thereof, e.g.,containing at least one asymmetric center. Exemplary chiral amino acidsinclude, but are not limited to, (L)-alanine, (D)-alanine, (L)-asparticacid, (D)-aspartic acid, (L)-phenylalanine, (D)-phenylalanine,(2S)-2-aminopentanedioic acid, (L)-asparagine, (D)-asparagine,(L)-cysteine, (D)-cysteine, (L)-glutamine, (D)-glutamine, (L)-glutamicacid, (D)-glutamic acid, (L)-proline, (D)-proline, (L)-selenocysteine,(D)-selenocysteine, (L)-serine, (D)-serine, (L)-tyrosine, (D)-tyrosine,(L)-arginine, (D)-arginine, (L)-histidine, (D)-histidine,(L)-isoleucine, (D)-isoleucine, (L)-leucine, (D)-leucine, (L)-lysine,(D)-lysine, (L)-methionine, (D)-methionine, (L)-threonine,(D)-threonine, (L)-tryptophan, (D)-tryptophan, (L)-valine, (D)-valine,(L)-ornithine, (D)-ornithine, (3R)-aminobutyrate, (3S)-aminobutyrate andpolypeptides thereof and/or salts thereof.

In certain embodiments, the solution is a buffered solution. In certainembodiments, the buffered solution is a sodium phosphate bufferedsolution.

In certain embodiments, the pH of the solution is between about 5 andabout 9, between about 5 and about 8, between about 6 and about 8,between about 7 and about 8, between about 7 and about 7.5, or betweenabout 7.5 and about 8.

In certain embodiments, the pH is of the solution is less than about 9,less than about 8.5, or less than about 8. In certain embodiments, thepH of the solution is about 7. In certain embodiments, the pH of thesolution is about 7.5. In certain embodiments, the pH of the solution isabout 8.

In certain embodiments, the compound of formula (I) or a salt thereofand a compound of formula (II) or a salt thereof are selected from anyset of compounds provided in Tables 1, 2, 3, 4 or 5.

In certain embodiments, the process further comprises contacting acompound of formula (II) or a salt thereof with a sulfonylating agent toprovide a compound of formula (III):

-   -   or a salt thereof,        wherein R²³ is alkyl or aryl.

In certain embodiments, the sulfonylating agent is selected frombenzenesulfonyl chloride, benzenesulfonyl anhydride, p-toluenesulfonylchloride, p-toluenesulfonyl anhydride, methanesulfonyl chloride, andmethanesulfonyl anhydride. In certain embodiments, the sulfonylatingagent is methanesulfonyl chloride or methanesulfonyl anhydride, and R²³is —CH₃.

The details of additional or alternative embodiments are set forth inthe accompanying Detailed Description and Examples as described below.Other features, objects, and advantages of the invention will beapparent from this description and from the claims.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987, each of which is incorporated hereinby reference.

Certain compounds of the present invention comprise one or moreasymmetric centers, and thus can exist in various isomeric forms, e.g.,enantiomers and/or diastereomers. The compounds provided herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer. Incertain embodiments, the compounds of the invention are enantiopurecompounds. In certain other embodiments, mixtures of stereoisomers areprovided.

Furthermore, certain compounds, as described herein may have one or moredouble bonds that can exist as either the cis or trans, or the E or Zisomer, unless otherwise indicated. The invention additionallyencompasses the compounds as individual isomers substantially free ofother isomers, and alternatively, as mixtures of various isomers, e.g.,racemic mixtures of E/Z isomers or mixtures enriched in one E/Z isomer.

The terms “enantiomerically enriched,” “enantiomerically pure” and“non-racemic,” as used interchangeably herein, refer to compositions inwhich the percent by weight of one enantiomer is greater than the amountof that one enantiomer in a control mixture of the racemic composition(e.g., greater than 1:1 by weight). For example, an enantiomericallyenriched preparation of the (S)-enantiomer, means a preparation of thecompound having greater than 50% by weight of the (S)-enantiomerrelative to the (R)-enantiomer, more preferably at least 75% by weight,and even more preferably at least 80% by weight. In some embodiments,the enrichment can be much greater than 80% by weight, providing a“substantially enantiomerically enriched,” “substantiallyenantiomerically pure” or a “substantially non-racemic” preparation,which refers to preparations of compositions which have at least 85% byweight of one enantiomer relative to other enantiomer, more preferablyat least 90% by weight, and even more preferably at least 95% by weight.Enantiomers can be isolated from mixtures by methods known to thoseskilled in the art, including chiral high pressure liquid chromatography(HPLC) and the formation and crystallization of chiral salts; orpreferred enantiomers can be prepared by asymmetric syntheses. See, forexample, Jacques, et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen, S. H., et al., Tetrahedron 33:2725(1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill,NY, 1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972), each of which is incorporated herein by reference.

Carbon atoms, unless otherwise specified, may optionally be substitutedwith one or more substituents. The number of substituents is typicallylimited by the number of available valences on the carbon atom, and maybe substituted by replacement of one or more of the hydrogen atoms thatwould be available on the unsubstituted group. Suitable substituents areknown in the art and include, but are not limited to, alkyl, alkenyl,alkynyl, alkoxy, alkoxy, alkylthio, aryl, aryloxy, arylthio, aralkyl,heteroaryl, heteroaralkyl, cycloalkyl, heterocyclyl, halo, azido,hydroxyl, thio, amino, nitro, nitrile, imido, amido, carboxylic acid,aldehyde, carbonyl, ester, silyl, haloalkyl, haloalkoxy (e.g.,perfluoroalkoxy such as —OCF₃), ═O, ═S, and the like.

When a range of values is listed, it is intended to encompass each valueand sub range within the range. For example, an alkyl group containing1-6 carbon atoms (C₁₋₆ alkyl) is intended to encompass, C₁, C₂, C₃, C₄,C₅, C₆, C₁₋₆, C₂₋₆, C₃₋₆, C₄₋₆, C₅₋₆, C₁₋₅, C₂₋₅, C₃₋₅, C₄₋₅, C₁₋₄,C₂₋₄, C₃₋₄, C₁₋₃, C₂₋₃, and C₁₋₂ alkyl.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radical containing between one and thirtycarbon atoms. In certain embodiments, the alkyl group contains 1-20carbon atoms. Alkyl groups, unless otherwise specified, may optionallybe substituted with one or more substituents. In certain embodiments,the alkyl group contains 1-10 carbon atoms. In certain embodiments, thealkyl group contains 1-6 carbon atoms. In certain embodiments, the alkylgroup contains 1-5 carbon atoms. In certain embodiments, the alkyl groupcontains 1-4 carbon atoms. In certain embodiments, the alkyl groupcontains 1-3 carbon atoms. In certain embodiments, the alkyl groupcontains 1-2 carbon atoms. In certain embodiments, the alkyl groupcontains 1 carbon atom. Examples of alkyl radicals include, but are notlimited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl,n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, andthe like.

The term “alkenyl,” as used herein, denotes a straight- orbranched-chain hydrocarbon radical having at least one carbon-carbondouble bond by the removal of a single hydrogen atom, and containingbetween two and thirty carbon atoms. Alkenyl groups, unless otherwisespecified, may optionally be substituted with one or more substituents.In certain embodiments, the alkenyl group contains 2-20 carbon atoms. Incertain embodiments, the alkenyl group contains 2-10 carbon atoms. Incertain embodiments, the alkenyl group contains 2-6 carbon atoms. Incertain embodiments, the alkenyl group contains 2-5 carbon atoms. Incertain embodiments, the alkenyl group contains 2-4 carbon atoms. Incertain embodiment, the alkenyl group contains 2-3 carbon atoms. Incertain embodiments, the alkenyl group contains 2 carbon atoms. Alkenylgroups include, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like.

The term “alkynyl,” as used herein, denotes a straight- orbranched-chain hydrocarbon radical having at least one carbon-carbontriple bond by the removal of a single hydrogen atom, and containingbetween two and thirty carbon atoms. Alkynyl groups, unless otherwisespecified, may optionally be substituted with one or more substituents.In certain embodiments, the alkynyl group contains 2-20 carbon atoms. Incertain embodiments, the alkynyl group contains 2-10 carbon atoms. Incertain embodiments, the alkynyl group contains 2-6 carbon atoms. Incertain embodiments, the alkynyl group contains 2-5 carbon atoms. Incertain embodiments, the alkynyl group contains 2-4 carbon atoms. Incertain embodiments, the alkynyl group contains 2-3 carbon atoms. Incertain embodiments, the alkynyl group contains 2 carbon atoms.Representative alkynyl groups include, but are not limited to, ethynyl,2-propynyl (propargyl), 1-propynyl, and the like.

The terms “cycloalkyl”, used alone or as part of a larger moiety, referto a saturated monocyclic or bicyclic hydrocarbon ring system havingfrom 3-15 carbon ring members. Cycloalkyl groups, unless otherwisespecified, may optionally be substituted with one or more substituents.In certain embodiments, cycloalkyl groups contain 3-10 carbon ringmembers. In certain embodiments, cycloalkyl groups contain 3-9 carbonring members. In certain embodiments, cycloalkyl groups contain 3-8carbon ring members. In certain embodiments, cycloalkyl groups contain3-7 carbon ring members. In certain embodiments, cycloalkyl groupscontain 3-6 carbon ring members. In certain embodiments, cycloalkylgroups contain 3-5 carbon ring members. Cycloalkyl groups include,without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. The term “cycloalkyl” also includessaturated hydrocarbon ring systems that are fused to one or more aryl orheteroaryl rings, such as decahydronaphthyl or tetrahydronaphthyl, wherethe point of attachment is on the saturated hydrocarbon ring.

The term “aryl” used alone or as part of a larger moiety (as in“aralkyl”), refers to an aromatic monocyclic and bicyclic hydrocarbonring system having a total of 6-10 carbon ring members. Aryl groups,unless otherwise specified, may optionally be substituted with one ormore substituents. In certain embodiments of the present invention,“aryl” refers to an aromatic ring system which includes, but not limitedto, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bearone or more substituents. Also included within the scope of the term“aryl”, as it is used herein, is a group in which an aryl ring is fusedto one or more nonaromatic rings, such as indanyl, phthalimidyl ortetrahydronaphthalyl, and the like, where the point of attachment is onthe aryl ring.

The term “aralkyl” refers to an alkyl group, as defined herein,substituted by aryl group, as defined herein, wherein the point ofattachment is on the alkyl group.

The term “heteroatom” refers to boron, phosphorus, selenium, nitrogen,oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur,and any quaternized form of abasic nitrogen.

The terms “heteroaryl” used alone or as part of a larger moiety, e.g.,“heteroaralkyl”, refer to an aromatic monocyclic or bicyclic hydrocarbonring system having 5-10 ring atoms wherein the ring atoms comprise, inaddition to carbon atoms, from one to five heteroatoms. Heteroarylgroups, unless otherwise specified, may optionally be substituted withone or more substituents. When used in reference to a ring atom of aheteroaryl group, the term “nitrogen” includes a substituted nitrogen.Heteroaryl groups include, without limitation, thienyl, furanyl,pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-”,as used herein, also include groups in which a heteroaryl ring is fusedto one or more aryl, cycloalkyl or heterocycloalkyl rings, wherein thepoint of attachment is on the heteroaryl ring. Nonlimiting examplesinclude indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,tetrahydroquinolinyl, and tetrahydroisoquinolinyl.

The term “heteroaralkyl” refers to an alkyl group, as defined herein,substituted by a heteroaryl group, as defined herein, wherein the pointof attachment is on the alkyl group.

As used herein, the terms “heterocycloalkyl” or “heterocyclyl” refer toa stable nonaromatic 5-7 membered monocyclic hydrocarbon or stablenonaromatic 7-10 membered bicyclic hydrocarbon that is either saturatedor partially unsaturated, and having, in addition to carbon atoms, oneor more heteroatoms. Heterocycloalkyl or heterocyclyl groups, unlessotherwise specified, may optionally be substituted with one or moresubstituents. When used in reference to a ring atom of aheterocycloalkyl group, the term “nitrogen” includes a substitutednitrogen. The point of attachment of a heterocycloalkyl group may be atany of its heteroatom or carbon ring atoms that results in a stablestructure. Examples of heterocycloalkyl groups include, withoutlimitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl,pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl,dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl,and quinuclidinyl. “Heterocycloalkyl” also include groups in which theheterocycloalkyl ring is fused to one or more aryl, heteroaryl orcycloalkyl rings, such as indolinyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocycloalkyl ring.

The term “unsaturated”, as used herein, means that a moiety has one ormore double or triple bonds.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups, such asaryl or heteroaryl moieties, as defined herein.

The term “diradical” as used herein refers to an alkyl, alkenyl,alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, andheteroaralkyl groups, as described herein, wherein 2 hydrogen atoms areremoved to form a divalent moiety. Diradicals are typically end with asuffix of “-ene”. For example, alkyl diradicals are referred to asalkylenes (for example:

and —(CR′₂)_(x)— wherein R′ is hydrogen or other substituent and x is 1,2, 3, 4, 5 or 6); alkenyl diradicals are referred to as “alkenylenes”;alkynyl diradicals are referred to as “alkynylenes”; aryl and aralkyldiradicals are referred to as “arylenes” and “aralkylenes”, respectively(for example:

heteroaryl and heteroaralkyl diradicals are referred to as“heteroarylenes” and “heteroaralkylenes”, respectively (for example:

cycloalkyl diradicals are referred to as “cycloalkylenes”;heterocycloalkyl diradicals are referred to as “heterocycloalkylenes”;and the like.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo,—Br), and iodine (iodo, —I).

As used herein, the term “haloalkyl” refers to an alkyl group, asdescribed herein, wherein one or more of the hydrogen atoms of the alkylgroup is replaced with one or more halogen atoms. In certainembodiments, the haloalkyl group is a perhaloalkyl group, that is,having all of the hydrogen atoms of the alkyl group replaced withhalogens (e.g., such as the perfluoroalkyl group —CF₃).

As used herein, the term “azido” refers to the group —N₃.

As used herein, the term “nitrile” refers to the group —CN.

As used herein, the term “nitro” refers to the group —NO₂.

As used herein, the term “hydroxyl” or “hydroxy” refers to the group—OH.

As used herein, the term “thiol” or “thio” refers to the group —SH.

As used herein, the term “carboxylic acid” refers to the group —CO₂H.

As used herein, the term “aldehyde” refers to the group —CHO.

As used herein, the term “alkoxy” refers to the group —OR′, wherein R′is an alkyl, alkenyl or alkynyl group, as defined herein.

As used herein, the term “aryloxy” refers to the group —OR′, whereineach R′ is an aryl or heteroaryl group, as defined herein.

As used herein, the term “alkylthio” or “alkylthiooxy” refers to thegroup —SR′, wherein each R′ is, independently, a carbon moiety, such as,for example, an alkyl, alkenyl, or alkynyl group, as defined herein.

As used herein, the term “arylthio” refers to the group —SR′, whereineach R′ is an aryl or heteroaryl group, as defined herein.

As used herein, the term “amino” refers to the group —NR′₂, wherein eachR′ is, independently, hydrogen, a carbon moiety, such as, for example,an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein,or two R′ groups together with the nitrogen atom to which they are boundform a 5-8 membered ring.

As used herein, the term “carbonyl” refers to the group —C(═O)R′,wherein R′ is, independently, a carbon moiety, such as, for example, analkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein.

As used herein, the term “ester” refers to the group —C(═O)OR′ or—OC(═O)R′ wherein each R′ is, independently, a carbon moiety, such as,for example, an alkyl, alkenyl, alkynyl, aryl or heteroaryl group, asdefined herein.

As used herein, the term “amide” or “amido” refers to the group—C(═O)N(R′)₂ or —NR′C(═O)R′ wherein each R′ is, independently, hydrogenor a carbon moiety, such as, for example, an alkyl, alkenyl, alkynyl,aryl or heteroaryl group, as defined herein, or two R′ groups togetherwith the nitrogen atom to which they are bound form a 5-8 membered ring.

As used herein, the term “imide” or “imido” refers to the group—C(═NR′)N(R′)₂ or —NR′C(═NR′)R′ wherein each R′ is,independently,hydrogen or a carbon moiety, such as, for example, analkyl, alkenyl, alkynyl, aryl or heteroaryl group, as defined herein, orwherein two R′ groups together with the nitrogen atom to which they arebound form a 5-8 membered ring.

As used herein “silyl” refers to the group —Si(R′)₃ wherein R′ is acarbon moiety, such as, for example, an alkyl, alkenyl, alkynyl, aryl orheteroaryl group.

The term “salt” refers to inorganic and organic acid addition salts ofcompounds of the present invention. Non-limiting examples ofrepresentative salts include salts derived from suitable inorganic andorganic acids, e.g., hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other acid addition salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxyethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike (see, for example, Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19, incorporated herein by reference).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical synthesis of IPI-926 in six steps from aketone starting material as described in Tremblay et al., “Discovery ofa Potent and Orally Active Hedgehog Pathway Antagonist (IPI-926)” J.Med. Chem. (2009) 52:4400-4418. The inventive transamination methodshortens this route by at least three steps.

FIG. 2 depicts two types of enzymatic transaminations. FIG. 2 a depictsthe Lalanine dehydrogenase (LADH)/formate dehydrogenase (FDH) promotedtransamination. FIG. 2 b depicts the lactate dehydrogenase (LDH)/Glucosedehydrogenase (GDH) promoted transamination.

DETAILED DESCRIPTION

Provided is a process for preparing a compound of formula (II):

or a salt thereof;

-   -   from a compound of formula (I):

or a salt thereof;

-   -   wherein:    -   R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl,        —OR¹⁶, —C(O)R¹⁶, —CO₂R¹⁶, —SO₂R¹⁶, —C(O)N(R¹⁷)(R¹⁷),        [C(R¹⁶)₂]_(q)—R¹⁶, —[(W)—N(R¹⁷)C(O)]_(q)R¹⁶, —[(W)—C(O)]_(q)R¹⁶,        —[(W)—C(O)]_(q)R¹⁶, —[(W)—OC(O)]_(q)R¹⁶, —[(W)—SO₂]_(q)R¹⁶,        —[(W)—N(R¹⁷)SO₂]_(q)R¹⁶, —[(W)—C(O)N(R¹⁷)]_(q)R¹⁷,        —[(W)—O]_(q)R¹⁶, —[(W)—N(R¹⁷)]_(q)R¹⁶, or —[(W)—S]_(q)R¹⁶;        wherein W is a diradical and q is 1, 2, 3, 4, 5, or 6;    -   each R² and R³ is, independently, H, alkyl, alkenyl, alkynyl,        aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl,        heteroaralkyl, haloalkyl, halo, —OR¹⁶, —OR¹⁶, —N(R¹⁷)₂, or        —SR¹⁶,    -   or R² and R³ taken together form a double bond or form a group

wherein Z is NR¹⁷, O, or C(R¹⁸)₂;

-   -   R⁴ is independently H, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶;    -   each R⁵ and R⁶, is, independently, H, alkyl, alkenyl, alkynyl,        aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl,        heteroaralkyl, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶;    -   or R⁵ and R⁶ taken together with the carbon to which they are        bonded, form C═O, C═S, C═N—OR¹⁷, C═N—R¹⁷, C═N—N(R′⁷)₂, or form        an optionally substituted 3-8 membered ring;    -   each R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ is, independently, H,        alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,        aralkyl, heteroaryl, heteroaralkyl, halo, —OR¹⁶, —N(R¹⁷)₂, or        —SR¹⁶;    -   or R¹⁰ and R¹¹ taken together, or R¹¹ and R¹² taken together,        form a double bond or form a group

wherein Z is NR¹⁷, O, or C(R¹⁸)₂;

-   -   each R¹⁴ and R¹⁵ is, independently, H, halo, —OR¹⁶, —N(R¹⁷)₂, or        —SR¹⁶;    -   or R¹⁴ and R¹⁵ taken together with the carbon to which they are        bonded, form C═O or C═S;    -   X is a bond or the group —C(R¹⁹)₂—; wherein each R¹⁹ is,        independently, H, alkyl, aralkyl, halo, —CN, —OR¹⁶, or —N(R¹⁷)₂;    -   R¹⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or        —[C(R²⁰)₂]_(p)—R²¹ wherein p is 0-6; or any two occurrences of        R¹⁶ on the same substituent are taken together to form a 4-8        membered optionally substituted ring;    -   R¹⁷ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁰,        —C(═O)OR²⁰, —SO₂R²⁰, —C(═O)N(R²⁰)₂, or —[C(R²⁰)₂]_(p)—R²¹        wherein p is 0-6; or any two occurrences of R¹⁷ on the same        substituent are taken together to form a 4-8 membered optionally        substituted ring;    -   R¹⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —CN,        —OR²⁰, —OSi(R²⁰)₃, —C(═O)R²⁰, —C(═O)OR²⁰, —SO₂R²⁰, or        —C(═O)N(R²⁰)₂;    -   R²⁰ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁰ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring;    -   R²¹ is —OR²², —N(R²²)C(═O)R²², —N(R²²)C(═O)OR²²,        —N(R²²)SO₂(R²²), —C(═O)R²²N(R²²)₂, —OC(═O)R²²N(R²²)(R²²),        —SO₂N(R²²)(R²²), —N(R²²)(R²²), —C(═O)OR²², —C(═O)N(OH)(R²²),        —OS(O)₂OR²², —S(O)₂OR²², —OP(═O)(OR²²)(OR²²),        —N(R²²)P(O)(OR²²)(OR²²), or —P(═O)(OR²²)(OR²²); and    -   R²² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl; or any two        occurrences of R²² on the same substituent are taken together to        form a 4-8 membered optionally substituted ring;    -   the process comprising contacting a compound of formula (I) or a        salt thereof, an amino donor molecule, and an amine transaminase        enzyme in a solution to provide a compound of formula (II) or a        salt thereof.

In certain embodiments, R¹ is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl,haloalkyl, —OR¹⁶, —C(O)R¹⁶, —CO₂R¹⁶, —SO₂R¹⁶, —C(O)N(R¹⁷)(R¹⁷), or—[C(R¹⁶)₂]_(q)—R¹⁶. In certain embodiments, R¹ is H, aralkyl, —C(O)R¹⁶,—CO₂R¹⁶, —SO₂R¹⁶ or —C(O)N(R¹⁷)(R¹⁷). In certain embodiments, R¹ is H,aralkyl or —CO₂R¹⁶.

In certain embodiments, R¹ is H.

In certain embodiments, R¹ is aralkyl.

In certain embodiments, R¹ is —CO₂R¹⁶. In certain embodiments, R¹ is—CO₂R¹⁶ and R¹⁶ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl or heteroaralkyl. In certainembodiments, R¹ is a -Boc group (e.g., wherein R¹ is —CO₂R¹⁶ and R¹⁶ ist-butyl). In certain embodiments, R¹ is a —CBz group (e.g., wherein R¹is —CO₂R¹⁶ and R¹⁶ is benzyl).

In certain embodiments, R² and R³ taken together form a double bond.

In certain embodiments, R² and R³ form a group:

-   -   wherein Z is —NR¹⁷—, —O—, or —C(R¹⁸)₂—. In certain embodiments,        Z is —C(R¹⁸)₂—. In certain embodiments, Z is —CH₂—.

In certain embodiments, X is a bond. For example, in certainembodiments, wherein R² and R³ are taken together form a double bond, orwherein R² and R³ form a group:

-   -   and Z is —NR¹⁷—, —O—, or —C(R¹⁸)₂—, then X is a bond.

In certain embodiments, X is the group —C(R¹⁹)₂—. In certainembodiments, R¹⁹ is H, e.g., wherein X is —CH₂—.

In certain embodiments, wherein R² and R³ are taken together form adouble bond, then X is the group —C(R¹⁹)₂—. In certain embodiments,wherein R² and R³ are taken together form a double bond, then X is thegroup —CH₂—.

In certain embodiments, R⁴ is H.

In certain embodiments, each R⁵ and R⁶, is, independently, H, or R⁵ andR⁶ taken together, along with the carbon to which they are bonded, formC═O. In certain embodiments, each of R⁵ and R⁶ is independently H. Incertain embodiments, R⁵ and R⁶ taken together with the carbon to whichthey are bonded form C═O.

In certain embodiments, R⁷ and R⁸ are each H.

In certain embodiments, R⁹ and R¹⁰ are each H.

In certain embodiments, R¹¹ is a H.

In certain embodiments, R¹² and R¹³ are each H.

In certain embodiments, R¹⁴ and R¹⁵ are each H.

In certain embodiments, each of R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴ andR¹⁵ is H.

In certain embodiments, R⁹ is H and R¹⁰ and R¹¹ taken together form adouble bond.

In certain embodiments, R¹³ is H, and R¹¹ and R¹² taken together form adouble bond.

In certain embodiments, the compound of formula (I) is a compound of theformula (I-AA):

-   -   or salt thereof,    -   and the compound of formula (II) is a compound of the formula        (II-AA):

-   -   or salt thereof,    -   wherein R¹, R², R³, R⁵, R⁶, R¹⁰, R¹¹, R¹² and X are as defined        herein.

In certain embodiments, the compound of formula (I) is a compound of theformula (I-BB):

-   -   or salt thereof,    -   and the compound of formula (II) is a compound of the formula        (II-BB):

-   -   or salt thereof,    -   wherein R¹, R², R³, R⁵, R⁶ and X are as defined herein.

In certain embodiments, the compound of formula (I) is a compound of theformula (I-CC):

-   -   or salt thereof,    -   and the compound of formula (II) is a compound of the formula        (II-CC):

-   -   or salt thereof,    -   wherein R¹ and X are as defined herein.

Exemplary compounds of formula (I) are provided in U.S. Pat. No.7,230,004, U.S. Pat. No. 7,407,967, U.S. Publication No. 20080293754,and U.S. Publication No. 20090012109, each of which is incorporatedherein by reference in their entirety.

In certain embodiments, the compound of formula (I) or a salt thereof,and a compound of formula (II) or a salt thereof, are selected from theset of compounds, or salts thereof, provided in Tables 1, 2, 3 and 4,and wherein R¹ is as defined above and herein:

TABLE 1 Compound of formula (I) Compound of formula (II)

  (I-a)

  (II-a)

  (I-b)

  (II-b)

  (I-c)

  (II-c)

  (I-d)

  (II-d)

  (I-e)

  (II-e)

  (I-f)

  (II-f)

TABLE 2 Compound of formula (I) Compound of formula (II)

  (I-g)

  (II-g)

  (I-h)

  (II-h)

  (I-i)

  (II-i)

  (I-j)

  (II-j)

  (I-k)

  (II-k)

  (I-l)

  (II-l)

TABLE 3 Compound of formula (I) Compound of formula (II)

  (I-m)

  (II-m)

  (I-n)

  (II-n)

  (I-o)

  (II-o)

  (I-p)

  (II-p)

  (I-q)

  (II-q)

  (I-r)

  (II-r)

TABLE 4 Compound of formula (I) Compound of formula (II)

  (I-s)

  (II-s)

  (I-t)

  (II-t)

  (I-u)

  (II-u)

  (I-v)

  (II-v)

  (I-w)

  (II-w)

  (I-x)

  (II-x)

In certain embodiments, R¹ is H.

In certain embodiments, R¹ is aralkyl.

In certain embodiments, R¹ is —CO₂R¹⁶. In certain embodiments, R¹ is—CO₂R¹⁶ and R¹⁶ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl or heteroaralkyl. In certainembodiments, R¹ is a -Boc group (e.g., wherein R¹ is —CO₂R¹⁶ and R¹⁶ ist-butyl). In certain embodiments, R¹ is a —CBz group (e.g., wherein R¹is —CO₂R¹⁶ and R¹⁶ is benzyl).

In certain embodiments, the process preferentially generates a compoundof formula (II), or a salt thereof, from a compound of formula (I), orsalt thereof, wherein the newly-formed amino group provided in formula(II) has (R) or (S) stereochemistry.

For example, in certain embodiments, the process preferentiallygenerates a compound of formula (II), or salt thereof, wherein thenewly-formed amino group has (S) stereochemistry, e.g., a compound ofthe formula (S)-(II):

-   -   or a salt thereof,    -   wherein R₁, R₂, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹. R¹², R¹³,        R¹⁴, R¹⁵ and X are as defined herein.

In certain embodiments, the process preferentially generates a compoundof formula (II), or salt thereof, wherein the newly-formed amino grouphas (R) stereochemistry, e.g., a compound of the formula (R)-(II):

-   -   or a salt thereof,    -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,        R¹⁴, R¹⁵ and X are as defined herein.

As used herein, “preferentially generates” refers to the production ofone stereoisomer of a compound of formula (II) in excess over the otherstereoisomer. In certain embodiments, the process preferentiallygenerates a compound of formula (II), or a salt thereof, wherein thenewly-formed amino group has (R) or (S) stereochemistry, in greater than40% diastereomeric excess (de), greater than 50% de, greater than 60%de, greater than 70% de, greater than 75% de, greater than 80% de,greater than 85% de, greater than 90% de, greater than 95% de, greaterthan 98% de, or greater than 99% de, as determined by HPLC.

In a preferred embodiment, the process preferentially generates acompound of formula (II), or salt thereof, wherein the newly-formedamino group has (R) stereochemistry.

For example, in certain embodiments, the compound of formula (I) is ofthe formula (I-AA):

-   -   or salt thereof,    -   and the compound of formula (II) is of the formula (R)-(II-AA):

-   -   or salt thereof,    -   wherein R¹, R², R³, R⁵, R⁶, R¹⁰, R¹¹, R¹² and X are as defined        herein.

In certain embodiments, the compound of formula (I) is of the formula(I-BB):

-   -   or salt thereof,    -   and the compound of formula (II) is of the formula (R)-(II-BB):

-   -   or salt thereof,    -   wherein R¹, R², R³, R⁵, R⁶ and X are as defined herein.

In certain embodiments, the compound of formula (I) is of the formula(I-CC):

-   -   or salt thereof,    -   and the compound of formula (II) is of the formula (R)-(II-CC):

-   -   or salt thereof,    -   wherein R¹ and X are as defined herein.

In another preferred embodiment, the compound of formulae (I) and (II)are selected from the set of compounds, or salts thereof, provided inTable 1.

In certain preferred embodiments, the process preferentially generates acompound of formula (II) of Table 1, or salt thereof, wherein thenewly-formed amino group has (R) stereochemistry.

For example, in certain embodiments, the compound of formulae (I) and(II) are selected from a set of compounds, or salts thereof, provided inTable 5, wherein the newly-formed amino group of the compound of formula(II) has (R) stereochemistry:

TABLE 5 Compound of formula (I) Compound of formula (II)

  (I-a)

  (R)-(II-a)

  (I-b)

  (R)-(II-b)

  (I-c)

  (R)-(II-c)

  (I-d)

  (R)-(II-d)

  (I-e)

  (R)-(II-e)

  (I-f)

  (R)-(II-f)

In certain embodiments, the compound of formula (I) is a compound offormula (I-a):

-   -   or salt thereof,    -   and the compound of formula (II) is a compound of formula        (R)-(II-a):

-   -   or a salt thereof,    -   wherein R¹ is as defined herein.

In certain embodiments, R¹ is H.

In certain embodiments, R¹ is aralkyl.

In certain embodiments, R¹ is —CO₂R¹⁶. In certain embodiments, R¹ is—CO₂R¹⁶ and R¹⁶ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl or heteroaralkyl. In certainembodiments, R¹ is a -Boc group (e.g., wherein R¹ is —CO₂R¹⁶ and R¹⁶ ist-butyl). In certain embodiments, R¹ is a —CBz group (e.g., wherein R¹is —CO₂R¹⁶ and R¹⁶ is benzyl).

Masked Ketones

In certain embodiments, the compound of formula (I) is a masked ketone.In this context, a “masked ketone” refers to a chemically modifiedcompound of formula (I) or salt thereof containing a functional groupwhich is transformed in situ (e.g., by hydrolysis) to the ketone.

Exemplary masked ketones include, but are not limited to, aminals andhemiaminals (see, for example, Vogel et al., J. Org. Chem. (2004)69:4487-4491; Reeder and Meyers, Tetrahedron Letters-(1999)40:3115-3118, each of which is incorporated herein by reference),acetals and hemiacetals (see, for example, Boyce et al., Bioorg. Med.Chem. Lett. (2008) 18:5771-5773, incorporated herein by reference),hydrates (see, for example, Silverman et al., J. Med. Chem. (1987)31:1566-1570, incorporated herein by reference), imines (see, forexample, Hine et al., J. Am. Chem. Soc. (1970) 92:5194-5199,incorporated herein by reference), oximes (see, for example, Sha et al.,J. Am. Chem. Soc. (2006) 128:9687-9692, incorporated herein byreference), thiocarbonyls (see, for example, Kalm, J. Chem. Soc. (1961)2925-2929, incorporated herein by reference), thioacetals andthiohemiacetals (see, for example Ogura et al., TetrahedronLetters-(1986) 27:3665-3668, incorporated herein by reference), enolethers (see for example, Manis and Rathke, J. Org. Chem. (1981)46:5348-5351, incorporated herein by reference), and salts thereof.

For example, provided is a process for preparing a compound of formula(II):

or a salt thereof;

-   -   from a masked ketone of a compound of formula (I):

or salt thereof;

-   -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,        R¹⁴, R¹⁵ and X are as defined herein;    -   the process comprising contacting a masked ketone of a compound        of formula (I) or a salt thereof, an amino donor molecule, and        an amine transaminase enzyme in a solution to provide a compound        of formula (II) or a salt thereof.

In certain embodiments, a compound of formula (I) is a masked ketonehaving the formula (I-DD):

-   -   or a salt thereof,    -   wherein:    -   R²⁴ and R²⁵ are selected from —OR²⁶, —SR²⁶, and —N(R²⁶)₂,        -   or R²⁴ and R²⁵ are taken together to form the group ═S,            ═N—R²⁶, or ═N—OR²⁶,        -   or R²⁴ is —OR²⁷ or —O(C═O)R²⁷ and R²⁵ and R⁸ or R²⁵ and R⁹            are taken together to form a bond;    -   R²⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁷,        —C(═O)OR²⁷ or —C(═O)N(R²⁸)₂, or any two occurrences of R²⁶ are        taken together to form a 4-8 membered optionally substituted        ring;    -   R²⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; and    -   R²⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁸ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a hydrate, an acetal or a hemiacetal.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a hydrate, an acetal or a hemiacetal of the formula (I-EE):

-   -   or a salt thereof,    -   wherein:    -   R²⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁷,        —C(═O)OR²⁷ or —C(═O)N(R²⁸)₂, or any two occurrences of R²⁶ are        taken together to form a 4-8 membered optionally substituted        ring;    -   R²⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; and    -   R²⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁸ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a aminal or hemiaminal.

In certain embodiments, a compound of formula (I-DD), or a salt thereof,is a aminal or hemiaminal of the formula (I-FF):

-   -   or a salt thereof,    -   wherein:    -   R²⁵ is —OR²⁶ or —N(R²⁶)₂;    -   R²⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁷,        —C(═O)OR²⁷ or —C(═O)N(R²⁸)₂, or any two occurrences of R²⁶ are        taken together to form a 4-8 membered optionally substituted        ring;    -   R²⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; and    -   R²⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁸ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a thioacetal or thiohemiacetal.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a thioacetal or thiohemiacetal of the formula (I-GG):

-   -   or a salt thereof,    -   wherein:    -   R²⁵ is —OR²⁶ or —SR²⁶;    -   R²⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁷,        —C(═O)OR²⁷ or —C(═O)N(R²⁸)₂, or any two occurrences of R²⁶ are        taken together to form a 4-8 membered optionally substituted        ring;    -   R²⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; and    -   R²⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁸ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is an imine.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is an imine of the formula (I-HH):

-   -   or a salt thereof,    -   wherein:    -   R²⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁷,        —C(═O)OR²⁷ or —C(═O)N(R²⁸)₂;    -   R²⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; and    -   R²⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁸ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is an oxime.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is an oxime of the formula (I-JJ):

-   -   or a salt thereof,    -   wherein:    -   R²⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁷,        —C(═O)OR²⁷ or —C(═O)N(R²⁸)₂;    -   R²⁷ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; and    -   R²⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁸ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a thiocarbonyl.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is a thiocarbonyl of the formula (I-KK):

-   -   or a salt thereof.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is an enol ether.

In certain embodiments, a compound of formula (I-DD) or a salt thereof,is an enol ether of the formulae (I-LL) or (I-MM):

-   -   or a mixture thereof and/or a salt thereof,    -   wherein R²⁴ is —OR²⁷ or —O(C═O)R²⁷ and R²⁷ is alkyl, alkenyl,        alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,        heteroaryl, or heteroaralkyl.

Amino Donor Molecule

An “amino donor molecule” is a compound having an —NH₂ group which,during the course of the reaction, is transferred to the compound offormula (I). Amino donor molecules include both amines and amino acids.

In certain embodiments, the amino donor molecule is an amine or saltthereof (e.g., a primary amine). Exemplary amine include, but are notlimited to, pyridoxamine, methylbenzylamine, 2-aminobutane, propylamine, isopropyl amine, 1,1,1-trifluoropropan-2-amine,1,1,1,3,3,3-hexafluoropropan-2-amine, benzyl amine, 2-amino/butanol,1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane,1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane,1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,2-aminopropanol, 1-amino-1 phenylbutane, 1-phenyl-2-aminobutane,1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl) 3-aminobutane, 1-amino-1-(2-naphthyl)ethane,cis-2-methylcyclopentanamine, trans-2-methylcyclopentanamine,cis-3-methylcyclopentanamine, trans-3-methylcyclopentanamine,cis-2-ethylcyclopentanamine, trans-2-ethylcyclopentanamine,cis-3-ethylcyclopentanamine, trans-3-ethylcyclopentanamine,cis-2-methylcyclohexanamine, trans-2-methylcyclohexanamine,cis-3-methylcyclohexanamine, trans-3-methylcyclohexanamine,cis-2-ethylcyclohexanamine, trans-2-ethylcyclohexanamine,cis-3-ethylcyclohexanamine, trans-3-ethylcyclohexanamine,1-aminotetralin, 2-aminotetralin, 2-amino-5-methoxytetralin,1-aminoindane, 2-aminoindane, 2-amino-1-propanol, cis-1-amino-2-indanol,trans-1-amino-2-indanol, 1-amino-6-hydroxyindanamine, taurine, and saltsthereof.

In certain embodiments, the amino donor molecule is an amino acid or apolypeptide thereof and/or a salt thereof. A polypeptide, as usedherein, refers to two or more amino acids joined by a peptide bond. Incertain embodiments, the polypeptide is a dipeptide (e.g., two aminoacids joined by a peptide bond).

In certain embodiments, the amino acid is selected from glycine,alanine, aspartic acid, phenylalanine, 2-aminopentanedioic acid,3-aminobutyrate, γ-aminobutyrate, β-alanine, asparagine, cysteine,glutamic acid, glutamine, proline, selenocysteine, serine, tyrosine,arginine, histidine, ornithine, isoleucine, leucine, lysine, methionine,threonine, tryptophan, valine, and polypeptides thereof and/or saltsthereof.

In certain embodiments, the amino donor molecule is a chiral amino donormolecule or a salt thereof, e.g., an amino donor molecule containing atleast one asymmetric center. In certain embodiments, the amino group(—NH₂) to be transferred is attached to a chiral carbon. In certainembodiments, the chiral carbon has (R)-stereochemistry. In certainembodiments, the chiral carbon has (S)-stereochemistry.

In certain embodiments, the chiral amino donor molecule is a chiralamine or a salt thereof, e.g., an amine containing at least oneasymmetric center. Exemplary chiral amines include, but are not limitedto, (R)-methylbenzylamine, (S)-methylbenzylamine, (S)-2-aminobutane,(R)-2-aminobutane, (S)-1-aminoindane, (R)-1-aminoindane,(R)-1,1,1-trifluoropropan-2-amine, (S)-1,1,1-trifluoropropan-2-amine,(R)-2-amino-1-propanol, (S)-2-amino-1-propanol,(1R,25)-cis-1-amino-2-indanol, (1R,2R)-trans-1-amino-2-indanol,1-(R)-amino-6-hydroxyindanamine, 1-(S)-amino-6-hydroxyindanamine,(R)-2-amino-1-butanol, (S)-2-amino-1-butanol,(R)-1-amino-1-phenylethane, (S)-1-amino-1-phenylethane,(R)-1-amino-1-(2-methoxy-5-fluorophenyl)ethane,(S)-1-amino-1-(2-methoxy-5-fluorophenyl)ethane,(R)-1-amino-1-phenylpropane, (S)-1-amino-1-phenylpropane,(R)-1-amino-1-(4-hydroxyphenyl)-propane,(S)-1-amino-1-(4-hydroxyphenyl)-propane,(R)-1-amino-1-(4-bromophenyl)propane,(S)-1-amino-1-(4-bromophenyl)propane,(R)-1-amino-1-(4-nitrophenyl)propane,(S)-1-amino-1-(4-nitrophenyl)propane, (R)-1-phenyl-2-aminopropane,(S)-1-phenyl-2-aminopropane,(R)-1-(3-trifluoromethylphenyl)-2-aminopropane,(S)-1-(3-trifluoromethylphenyl)-2-aminopropane(R)-1-amino-1-phenylbutane, (S)-1-amino-1-phenylbutane,(R)-1-phenyl-2-aminobutane, (S)-1-phenyl-2-aminobutane,(R)-1-(2,5-di-methoxy-4-methylphenyl)-2-aminobutane,(S)-1-(2,5-di-methoxy-4-methylphenyl)-2-aminobutane,(R)-1-phenyl-3-aminobutane, (S)-1-phenyl-3-aminobutane,(R)-1-(4-hydroxyphenyl)-3-aminobutane,(S)-1-(4-hydroxyphenyl)-3-aminobutane, (R)-1-amino-1-(2-naphthyl)ethane,(S)-1-amino-1-(2-naphthyl)ethane (R)-1-aminotetralin,(S)-1-aminotetralin, (R)-2-aminotetralin, (S)-2-aminotetralin,(R)-2-amino-5-methoxytetralin, (S)-2-amino-5-methoxytetralin,(1R,2S)-cis-2-methylcyclopentanamine,(1S,2R)-cis-2-methylcyclopentanamine,(1R,2R)-trans-2-methylcyclopentanamine,(1S,2S)-trans-2-methylcyclopentanamine,(1R,3S)-cis-3-methylcyclopentanamine,(1S,3R)-cis-3-methylcyclopentanamine,(1R,3R)-trans-3-methylcyclopentanamine,(1S,3S)-trans-3-methylcyclopentanamine,(1R,2S)-cis-2-ethylcyclopentanamine,(1S,2R)-cis-2-ethylcyclopentanamine,(1R,2R)-trans-2-ethylcyclopentanamine,(1S,2S)-trans-2-ethylcyclopentanamine,(1R,3S)-cis-3-ethylcyclopentanamine,(1S,3R)-cis-3-ethylcyclopentanamine,(1R,3R)-trans-3-ethylcyclopentanamine,(1S,3S)-trans-3-ethylcyclopentanamine,(1R,2S)-cis-2-methylcyclohexanamine,(1S,2R)-cis-2-methylcyclohexanamine,(1R,2R)-trans-2-methylcyclohexanamine,(1S,2S)-trans-2-methylcyclohexanamine,(1R,3S)-cis-3-methylcyclohexanamine,(1S,3R)-cis-3-methylcyclohexanamine,(1R,3R)-trans-3-methylcyclohexanamine,(1S,3S)-trans-3-methylcyclohexanamine,(1R,2S)-cis-2-ethylcyclohexanamine, (1S,2R)-cis-2-ethylcyclohexanamine,(1R,2R)-trans-2-ethylcyclohexanamine,(1S,2S)-trans-2-ethylcyclohexanamine,(1R,3S)-cis-3-ethylcyclohexanamine, (1S,3R)-cis-3-ethylcyclohexanamine,(1R,3R)-trans-3-ethylcyclohexanamine,(1S,3S)-trans-3-ethylcyclohexanamine, and salts thereof.

In certain embodiments, the chiral amino donor molecule is a chiralamino acid or a polypeptide thereof and/or a salt thereof, e.g.,containing at least one asymmetric center. Exemplary chiral amino acidsinclude, but are not limited to, (L)-alanine, (D)-alanine, (L)-asparticacid, (D)-aspartic acid, (L)-phenylalanine, (D)-phenylalanine,(2S)-2-aminopentanedioic acid, (L)-asparagine, (D)-asparagine,(L)-cysteine, (D)-cysteine, (L)-glutamine, (D)-glutamine, (L)-glutamicacid, (D)-glutamic acid, (L)-proline, (D)-proline, (L)-selenocysteine,(D)-selenocysteine, (L)-serine, (D)-serine, (L)-tyrosine, (D)-tyrosine,(L)-arginine, (D)-arginine, (L)-histidine, (D)-histidine,(L)-isoleucine, (D)-isoleucine, (L)-leucine, (D)-leucine, (L)-lysine,(D)-lysine, (L)-methionine, (D)-methionine, (L)-threonine,(D)-threonine, (L)-tryptophan, (D)-tryptophan, (L)-valine, (D)-valine,(L)-ornithine, (D)-ornithine, (3R)-aminobutyrate, (3S)-aminobutyrate andpolypeptides thereof and/or salts thereof.

In certain embodiments, the chiral amino donor molecule is(R)-methylbenzylamine or a salt thereof. In other embodiments, thechiral amino donor molecule is (S)-methylbenzylamine or a salt thereof.

In certain embodiments, the chiral amino donor molecule is (L)-alanineor a salt thereof. In certain embodiments, the chiral amino donormolecule is (D)-alanine or a salt thereof.

In certain embodiments, the chiral amino donor molecule is(S)-1-aminoindane. In certain embodiments, the chiral amino donormolecule is (R)-1-aminoindane.

In certain embodiments, the process comprises contacting a compound offormula (I), a chiral amino donor molecule, and an amine transaminaseenzyme in a solution to provide a compound of formula (II), or a saltthereof, with the newly-formed amino group having (S) stereochemistry.

In certain embodiments, the process comprises contacting a compound offormula (I), or a salt thereof, a chiral amino donor molecule, and anamine transaminase enzyme in a solution to provide a compound of formula(II), or a salt thereof, with the newly-formed amino group having (R)stereochemistry.

Amine Transaminase (ATA) Enzyme

An amine transaminase (ATA) enzyme catalyzes the transfer of the —NH₂group from the amino donor molecule to a compound having a ketonefunctional group, e.g., a compound of formula (I), in order to provide acompound of formula (II).

In certain embodiments, the amine transaminase enzyme preferentiallygenerates a compound of formula (II), or a salt thereof, with thenewly-formed amino group having (R) or (S) stereochemistry. As usedherein, “preferentially generates” refers to the production of onestereoisomer of a compound of formula (II) in excess over the otherstereoisomer.

In certain embodiments, the amine transaminase enzyme preferentiallygenerates a compound of formula (II), or a salt thereof, with thenewly-formed amino group having (R) stereochemistry.

In certain embodiments, the amine transaminase enzyme preferentiallygenerates a compound of formula (II), or a salt thereof, with thenewly-formed amino group having (S) stereochemistry.

In certain embodiments, the amine transaminase enzyme preferentiallygenerates a compound of formula (II), or a salt thereof, with thenewly-formed amino group having (R) or (S) stereochemistry, in greaterthan 40% diastereomeric excess (de), greater than 50% de, greater than60% de, greater than 70% de, greater than 75% de, greater than 80% de,greater than 85% de, greater than 90% de, greater than 95% de, greaterthan 98% de, or greater than 99% de, as determined by HPLC.

In certain embodiments, the amine transaminase enzyme preferentiallygenerates an enantiomerically pure compound of formula (II), or saltthereof.

In certain embodiments, the amine transaminase enzyme is an omega aminetransaminase enzyme, a broad-range transaminase, a glutamate-pyruvatetransaminase or a glutamate-oxaloacetic transaminase.

In certain embodiments, the amine transaminase enzyme is an omega aminetransaminase enzyme.

Exemplary omega amine transaminase enzymes include, but are not limitedto, omega amine transaminase enzymes from Codexis, Inc. (Redwood City,Calif.), such as ATA-101, ATA-102, ATA-103, ATA-104, ATA-105, ATA-106,ATA-107, ATA-108, ATA-109, ATA-110, ATA-113, ATA-114, ATA-115, ATA-116,ATA-117 and ATA-124; omega amine transaminases from Vibrio fluvialis,Alcaligenes denitrificans, Klebsiella pneumoniae, or Bacillusthuringiensis, such as is described in WO 2007093372, incorporatedherein by reference; omega amine transaminases from Chromobacteriumviolaceum, such as is described in Smithies et al., TetrahedronAsymmetry (2009) 570-574, incorporated herein by reference, omega aminetransaminases from Bacillus cereus, such as is described in Nakano etal., J. Biochem. (1977) 81:1375-1381, incorporated herein by reference,and omega amine transaminases from Arthrobactercitreus, such asdescribed in Cassimjee et al., ChemComm (2010) 46:5569-5571,incorporated herein by reference.

Other suitable exemplary omega amine transaminases which may be usedaccording to the present invention are described in Iwa-saki et al.,Biotechnol. Lett. (2003) 25:1843-1846; Shin et al., Biotechnol. Bioeng.(1997) 55:348-358; Shin and Kim, Biosc. Biotechnol. Biochem. (2001)65:1782-1788; Koszelewski et al., Trends in Biotechnology (2010)28:324-332, and Shin and Kim, Biotechnol. Bioeng. (1998) 60:534-540,each of which is incorporated herein by reference.

Immobilization of the amine transaminase enzyme can also be effective toimprove the stability of the enzyme which in turn will allow its re-use,thereby making the process more economical. Immobilization of theenzymes has been achieved by a simple adsorption onto a hydrophobicresin or by intermolecular covalent cross-linking of enzymes with avariety of functional groups or finally by incorporating enzymes intothe lattice of a polymer matrix or a membrane. Covalent immobilizationof the omega amine transaminase from Vibrio fluvialis JS17 has beenreported by Lee and co-workers where the authors adsorb the enzyme onchitosan beads and subsequently cross linked with glutaraldehyde (Yi etal., Proc. Biochem. (2007) 42:895-898, incorporated herein byreference). The immobilized amine transaminase enzyme on chitosan beadsretained ca. 77% of its activity after five consecutive reactions withthe substrate indicating the utility of the process.

In certain embodiments, the omega amine transaminase enzyme is an aminetransaminase enzyme from Codexis, Inc.

In certain embodiments, the omega amine transaminase enzyme is ATA-113.

In certain embodiments, the omega amine transaminase enzyme is ATA-117.

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Vibrio fluvialis

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Alcaligenes denitrificans.

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Arthrobactercitreus.

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Klebsiella pneumoniae.

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Bacillus thuringiensis.

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Bacillus cereus.

In certain embodiments, the omega amine transaminase enzyme is an omegaamine transaminase from Chromobacterium violaceum.

In certain embodiments, the omega amine transaminase enzyme is animmobilized omega amine transaminase enzyme.

Co-factors

In certain embodiments, the process further comprises adding a co-factorto the solution. Co-factors include prosthetic groups which are bound toan enzyme during the enzymatic reaction, and coenzymes which act totransfer chemical groups during the enzymatic reaction.

Exemplary co-factors include the prosthetic group pyridoxal phosphate(PLP) and coenzymes such as Lalanine dehydrogenase (LADH), lactatedehydrogenase (LDH), nicotinamide adenine dinucleotide phosphate(NADPH), nicotinamide adenine dinucleotide

(NAD), formate dehydrogenase (FDH) and glucose dehydrogenase (GDH).

In certain embodiments, the process further comprises adding theco-factor pyridoxal phosphate (PLP) to the solution.

In certain embodiments, the amine transaminase enzyme and the co-factorpyridoxal phosphate added to the solution are pre-complexed beforecontacting with the compound of formula I. In other embodiments, theamine transaminase (ATA) enzyme and pyridoxal phosphate added to thesolution are not pre-complexed before contacting with the compound offormula I (i.e., each is individually added to the solution).

In certain embodiments, the process further comprises adding a coenzymeto the solution. In certain embodiments, the process further comprisesadding one or more coenzymes selected from Lalanine dehydrogenase(LADH), lactate dehydrogenase (LDH), nicotinamide adenine dinucleotidephosphate (NADPH), nicotinamide adenine dinucleotide (NAD), formatedehydrogenase (FDH), and glucose dehydrogenase (GDH) to the solution.

In certain embodiments, the process further comprises adding theco-enzyme LADH to the solution.

In certain embodiments, the process further comprises adding theco-enzyme FDH to the solution.

In certain embodiments, the process further comprises adding theco-enzyme NAD to the solution.

In certain embodiments, the process further comprises adding theco-enzyme LDH to the solution.

In certain embodiments, the process further comprises adding theco-enzyme GDH to the solution.

In certain embodiments, the process further comprises adding a mixtureof LADH, FDH and NAD to the solution.

In certain embodiments, the process further comprises adding a mixtureof co-enzymes LDH, GDH and NAD to the solution.

In certain embodiments, the process further comprises adding a sugar tothe solution. In certain embodiments, the sugar is glucose.

In certain embodiments, process further comprises adding a pyruvatereductase mix to the solution. In certain embodiments, the processfurther comprises adding a mixture of co-enzymes LDH, GDH, NAD, and thesugar glucose (e.g., for example, pyruvate reductase mix PRM-102,available from Codexis, Inc.) to the solution.

In certain embodiments, the process further comprises adding ammonia oran ammonium salt to the solution. In certain embodiments, the ammoniumsalt is ammonium formate (NH₄CO₂H). Ammonium formate can be obtained insitu from the combination of formic acid and ammonia.

Other Reaction Conditions

In certain embodiments, the solution comprises an aqueous solution.

In certain embodiments, the aqueous solution is a buffered aqueoussolution. Exemplary buffers include, but are not limited to,3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),N,N-bis(2-hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)methylamine(Tris), N-tris(hydroxymethyl)methylglycine (Tricine),4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES),2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinic acid(Cacodylate), 2-(N-morpholino)ethanesulfonic acid (MES), carbonic acid,phosphate buffered saline (PBS), acetate, sodium phosphate, and saltsthereof.

In certain embodiments, the buffered aqueous solution is a sodiumphosphate buffered solution.

In certain embodiments, the solution further comprises a co-solvent. Incertain embodiments, the co-solvent is an organic solvent.

In certain embodiments, the organic solvent is water miscible. Incertain embodiments, the organic solvent is water immicible.

In certain embodiments, the solution is a monophasic system, e.g.,comprising an aqueous solution and one or more water miscible organicsolvents. Suitable water miscible organic solvents include, but are notlimited to, organic alcohols (e.g., methanol (MeOH), ethanol (EtOH),isopropanol (iPrOH) and 2,2,2-trifluoroethanol (CF₃CH₂OH)),dimethylsulfoxide (DMSO), dimethylformamide (DMF), glycols (e.g.,ethylene glycol and propylene glycol), and mixtures thereof.

In certain embodiments, the solution comprises an aqueous solution andan organic alcohol. In certain embodiments, the solution comprises anaqueous solution and methanol.

However, in other embodiments, the solution is a biphasic system, e.g.,comprising an aqueous solution and one or more water immicible organicsolvents. Suitable water immicible organic solvents include, but are notlimited to, alkanes (e.g., hexane, heptane, perfluorohexane), esters(e.g., ethyl acetate (EtOAc), isopropyl acetate (iPrOAc)), ketones(e.g., cyclohexanone), ethers (e.g., 2-methyl tetrahydrofuran), andaromatic hydrocarbons (e.g., toluene, xylenes, benzene).

In certain embodiments, the pH of the solution is between about 5 andabout 9, between about 5 and about 8, between about 6 and about 8,between about 7 and about 8, between about 7 and about 7.5, or betweenabout 7.5 and about 8.

In certain embodiments, the pH is of the solution is less than about 9,less than about 8.5, or less than about 8. In certain embodiments, thepH of the solution is about 7. In certain embodiments, the pH of thesolution is about 7.5. In certain embodiments, the pH of the solution isabout 8.

In certain embodiments, the temperature of the solution is at leastabout 20° C., at least about 25° C., at least about 30° C., or at leastabout 35° C. In certain embodiments, the temperature of the solution isbetween about 20° C. and about 50° C.

In certain embodiments, the process further comprises a resin.Adsorption of the ketone starting material (I) or the product amine (II)to the resin reduces their respective concentration in the reactionmedium, and thus reduces their propensity to inhibit the enzyme.Exemplary resins include, but are not limited to, Amberlite™, Amberlyst™and Dowex™ resins.

In certain embodiments, the process further comprises a solublizer suchas a cyclodextrin or a surfactant. Exemplary cyclodextrins include, butare not limited to, β-cyclodextrins and γ-cyclodextrins. Exemplarysurfactants include, but are not limited to sorbitan fatty acid esters(e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylenesorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80],sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60],sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate[Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate[Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylatedcastor oil, polyoxymethylene stearate, and Solutol), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), diethylene glycolmonolaurate, triethanolamine oleate, sodium oleate, potassium oleate,ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PluronicF 68, Poloxamer 188, and/or combinations thereof.

In certain embodiments, the process further comprises a sulfate (e.g.,for example, sodium bisulfate). For example, when benzylamine is used asthe amino donor molecule, sodium bisulfate reacts with the by-productbenzaldehyde to form an insoluble bisulfite adduct.

In certain embodiments, the process further comprises a dehydrogenaseenzyme (e.g., a yeast alcohol dehydrogenase (YADH) such as fromSaccharomyces cerevisiae). For example, when isopropyl amine is used asthe amino donor molecule, a YADH enzyme converts the acetone by-productto isopropanol, thereby shifting the equilibrium and driving thereaction to completion (see Cassimjee et al., Chem Comm (2010)46:5569-5571, incorporated herein by reference).

Additional Embodiments

In certain embodiments, provided is a process for preparing a compoundof formula (R)-(II-a):

or a salt thereof;

-   -   from a compound of formula (I-a):

or a salt thereof;

-   -   wherein:    -   R¹ is H, aralkyl, or CO₂R¹⁶;    -   R¹⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or        —[C(R²⁰)₂]_(p)—R²¹ wherein p is 0-6;    -   R²⁰ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any        two occurrences of R²⁰ on the same substituent are taken        together to form a 4-8 membered optionally substituted ring;    -   R²¹ is —OR²², —N(R²²)C(═O)R²², —N(R²²)C(═O)OR²²,        —N(R²²)SO₂(R²²), —C(═O)R²²N(R²²)₂, —OC(═O)R²²N(R²²)(R²²),        —SO₂N(R²²)(R²²), —N(R²²)(R²²), —C(═O)OR²², —C(═O)N(OH)(R²²),        —OS(O)₂OR²², —S(O)₂OR²², —OP(═O)(OR²²)(OR²²),        —N(R²²)P(O)(OR²²)(OR²²), or —P(═O)(OR²²)(OR²²); and    -   R²² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,        heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl; or any two        occurrences of R²² on the same substituent are taken together to        form a 4-8 membered optionally substituted ring;    -   the process comprising contacting a compound of formula (I-a) or        a salt thereof, an amino donor molecule, and an amine        transaminase enzyme in a solution to provide a compound of        formula (R)-(II-a) or a salt thereof.

In certain embodiments, R¹ is H, aralkyl, or —CO₂R¹⁶.

In certain embodiments, R¹ is H.

In certain embodiments, R¹ is aralkyl.

In certain embodiments, R¹ is —CO₂R¹⁶. In certain embodiments, R¹ is—CO₂R¹⁶ and R¹⁶ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl or heteroaralkyl. In certainembodiments, R¹ is a -Boc group (e.g., wherein R¹ is —CO₂R¹⁶ and R¹⁶ ist-butyl). In certain embodiments, R¹ is a —CBz group (e.g., wherein R¹is —CO₂R¹⁶ and R¹⁶ is benzyl).

In certain embodiments, the amino donor molecule is a chiral amino donormolecule. In certain embodiments, the chiral amino donor molecule is(R)-methylbenzylamine or a salt thereof. In other embodiments, thechiral amino donor molecule is (S)-methylbenzylamine or a salt thereof.

In certain embodiments, the chiral amino donor molecule is (L)-alanineor a salt thereof. In certain embodiments, the chiral amino donormolecule is (D)-alanine or a salt thereof.

In certain embodiments, the amine transaminase enzyme is an omega aminetransaminase enzyme. In certain embodiments, the omega aminetransaminase enzyme is ATA-113 from Codexis, Inc. In certainembodiments, the omega amine transaminase enzyme is ATA-117 fromCodexis, Inc. In certain embodiments, the omega amine transaminaseenzyme is an omega amine transaminase from Vibrio fluvialis.

Additional Steps

In certain embodiments, the process further comprises contacting acompound of formula (II):

-   -   or a salt thereof,    -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,        R¹⁴, R¹⁵ and X are as defined herein,    -   with a sulfonylating agent to provide a compound of formula        (III):

-   -   or a salt thereof,    -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,        R¹⁴, R¹⁵ and X are as defined herein, and    -   R²³ is alkyl or aryl.

Exemplary sulfonylating agents include, but are not limited to,benzenesulfonyl chloride, benzenesulfonyl anhydride, p-toluenesulfonylchloride, p-toluenesulfonyl anhydride, methanesulfonyl chloride, andmethanesulfonyl anhydride.

In certain embodiments, the sulfonylating agent is benzenesulfonylchloride or benzenesulfonyl anhydride, and R²³ is benzenyl (i.e.,—C₆H₅).

In certain embodiments, the sulfonylating agent is p-toluenesulfonylchloride or p-toluenesulfonyl anhydride, and R²³ is toluenyl (i.e.,—C₆H₄(p-CH₃)).

In certain embodiments, the sulfonylating agent is methanesulfonylchloride or methanesulfonyl anhydride, and R²³ is methyl (i.e., —CH₃).

In certain embodiments, the compound of formula (II) is:

or a salt thereof,

-   -   and the compound of formula (III) is:

-   -   or a salt thereof,    -   wherein R¹ and R²³ are as defined herein.

In certain embodiments, the compound of formula (II) is:

-   -   or a salt thereof,    -   and the compound of formula (III) is:

-   -   or a salt thereof,    -   wherein R¹ and R²³ are as defined herein.

In certain embodiments, R¹ is H.

In certain embodiments, R¹ is aralkyl.

In certain embodiments, R¹ is —CO₂R¹⁶.

In certain embodiments wherein R¹ is aralkyl or —CO₂R¹⁶, the processfurther comprises deprotecting the compound of formula (III) wherein R¹is aralkyl or —CO₂R¹⁶ to provide a compound of formula (III) wherein R¹is H. Exemplary deprotection methods include, but are not limited to,reducing conditions, such as hydrogenation.

For example, in certain embodiments, the process further comprisesdeprotecting the compound of formula (R)-(III-a):

-   -   or salt thereof,    -   wherein R¹ is aralkyl or —CO₂R¹⁶,    -   to provide a compound of formula (R)-(III-b):

or salt thereof,

-   -   wherein R²³ is as defined herein.

EXEMPLIFICATION

The present disclosure now being generally described, it will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration and are not intended tolimit the disclosure herein.

Enzymatic Transamination of Compound (I-a)

Materials and Methods

Enzymes.

Amine transaminase enzymes were purchased from commercially availablesources, stored at −20° C., and used as received: ATA-113 (Codexis,Redwood City, Calif.; Lot no. 104020902); ATA-117 (Codexis, RedwoodCity, Calif.; Lot no.104020902); omega-transaminase from Vibriofluvialis (Fluka; cat. no 08374); glutamate pyruvate transaminase(Fluka); broad range transaminase (Fluka).

Co-Enzymes.

Co-enzymes utilized during the investigation include: L-alaninedehydrogenase (LADH, Sigma, no. A7653-100U), formate dehydrogenase (FDH,Codexis, FDH-101) and pyruvate reductase mix (PRM-102, Codexis), whichis a mixture lactate dehydrogenase (LDH), glucose dehydrogenase (GDH),glucose and NAD⁺.

pH.

The following buffers were used during the investigation: 100 mM sodiumphosphate buffer (pH 7; Fluka no. 82637); 20 mM sodium phosphate buffer(pH 7.5; Fluka, no. 82592); 20 mM sodium phosphate buffer (pH 8; Fluka,no. 82593).

HPLC Method 1.

Symmetry C18 column 4.6×150 mm; flow rate 1.5 mL/min; mobile phaseA=0.1% TFA in water; mobile phase B=0.1% TFA in acetonitrile; 10 μLinjection; 40° C. column temperature; detection wavelength=215 nm (allspecies). Retention time of compound (II-a) (R¹═H: S-(II-a)=5.9 min;R-(II-a), =6.8 min).

HPLC Method 1 Gradient:

Time (minutes) % mobile phase A % mobile phase B 0 90 10 1.0 90 10 10.040 60 11.0 5 95 12.0 5 95 13.0 90 10 15.0 90 10

HPLC Method 2.

XBridge C8 column 4.6×75 mm; flow rate 1.0 mL/min; mobile phase A=10 mMammonium formate buffer, pH 3.8; mobile phase B=0.05% formic acid inacetonitrile; 5 μL injection; 40° C. column temperature; detectionwavelength=215 nm (all species). Retention time of compound (II-a)(R¹=Bn: S-(II-a)=7.0 min; R-(II-a)=7.5 min).

HPLC Method 2 Gradient:

Time (minutes) % mobile phase A % mobile phase B 0 80 20 2 80 20 16 5 9518 5 95 18.1 80 20 20 80 20

HPLC Method 3.

Eclipse XDB-C8 column 4.6×50 mm; flow rate 1.5 mL/min; mobile phaseA=0.1% TFA in water; mobile phase B=0.1% TFA in acetonitrile; 10 μLinjection; 40° C. column temperature; detection wavelength=215 nm (allspecies). Retention time of compound (II-a) (R¹=Cbz: S-(II-a)=6.0 min;R(II-a)=6.1 min).

HPLC Method 3 Gradient:

Time (minutes) % mobile phase A % mobile phase B 0 70 30 2.0 70 30 8.0 0100 10.0 0 100 12.0 70 30 15.0 70 30

General Experimental Methods

Method A:

Compound (I-a), a sodium phosphate buffered solution, 1 mM pyridoxalphosphate (PLP), an amine transaminase enzyme, an amino donor molecule,PRM-102 and, optionally, a co-solvent, were added to a vial. The vialwas capped and the reaction stirred at 30° C. for 72 hours. The reactionmixture was then lyophilized and the residue was re-dissolved inmethanol (2 mL). A 100 μL portion of the methanol solution was filteredand analyzed by HPLC. The results of Experiments 1-8 using Method A aresummarized in Table 6.

Method B:

Compound (I-a), a sodium phosphate buffered solution, 1 mM pyridoxalphosphate (PLP), amine transaminase enzyme, D-alanine, ammonium formate(NH₄CO₂H), L-alanine dehydrogenase (LADH) solution (8.05 U/mL), formatedehydrogenase (FDH) solution (10 U/mL), NAD (1 mM) and a co-solvent wereadded to a vial. The vial was capped and the reaction stirred at 30° C.for 72 hours. The reaction mixture was then lyophilized and the residuewas redissolved in methanol (2 mL). A 100 μL portion of the methanolsolution was filtered and analyzed by HPLC. The results of Experiments1-10 using Method B are summarized in Table 7.

Method C:

Compound (I-a), a sodium phosphate buffered solution, pyridoxalphosphate (PLP) (0.5 mM to 1 mM), an amine transaminase enzyme, an aminodonor molecule, and, optionally, a co-solvent and PRM-102, were added toa vial. The vial was capped and the reaction stirred at 37° C. for 24hours to about 6 days (144 hours). The reaction mixture was thenlyophilized and the residue was redissolved in methanol (2 mL). A 100 μLportion of the methanol solution was filtered and analyzed by HPLC. Theresults of Experiments 1-28 using Method C are summarized in Table 8.

Method D:

Compound (I-a), a sodium phosphate buffered solution, pyridoxalphosphate (PLP) (0.5 mM), an amine transaminase enzyme, an amino donormolecule (5 equivalents), PRM-102, and, optionally, a co-solvent, wereadded to a vial. The vial was capped and the reaction stirred at 37° C.for 24 hours to about 7 days (168 hours). A 100 μL portion of thereaction mixture was diluted with 1 volume of MeOH and analyzed by HPLC.The results of Experiments 1-13 using Method D are summarized in Table9.

Method E:

Compound (I-a), a sodium phosphate buffered solution, pyridoxalphosphate (PLP) (0.5 mM), an amine transaminase enzyme, L-alanine (5equivalents), PRM-102, and methanol (10% v/v) were added to a vial. Thevial was capped and the reaction stirred at 37° C. for 24 hours to about7 days (168 hours). A 100 μL portion of the reaction mixture was dilutedwith 1 volume of MeOH and analyzed by HPLC. The results of Experiments1-5 using Method E are summarized in Table 10.

Method F:

Compound (I-a), a sodium phosphate buffered solution (pH=7.5), pyridoxalphosphate (PLP) (0.5 mM), an amine transaminase enzyme, L-alanine (5equivalents), PRM-102, and methanol (10% v/v) were added to a vial. Thevial was capped and the reaction stirred at 37° C. for 24 hours to about7 days (168 hours). The pH of the aqueous layer was monitored at theonset and during the course of the reaction and if necessary, it wasreadjusted to 7.5 using 0.1 M NaOH solution. A 100 μL portion of thereaction mixture was diluted with 1 volume of MeOH and analyzed by HPLC.The results of Experiments 1-9 using Method F are summarized in Table11.

Method G:

Compound (I-a), a sodium phosphate buffered solution (pH=7.5), pyridoxalphosphate (PLP) (0.5 mM), an amine transaminase enzyme, L-alanine (5equivalents), PRM-102, an additive (100% wt/wt) and methanol (10% v/v)were added to a vial. The vial was capped and the reaction stirred at37° C. for 24 hours to about 7 days (168 hours). A 100 μL portion of thereaction mixture was diluted with 1 volume of MeOH and analyzed by HPLC.The results of Experiments 1-5 using Method G are summarized in Table12.

Method H:

Compound (I-a), a sodium phosphate buffered solution (pH=7.5), pyridoxalphosphate (PLP) (0.5 mM), an amine transaminase enzyme, L-alanine (5equivalents), PRM-102, a co-solvent (10 to 20% v/v) were added to avial. The vial was capped and the reaction stirred at 37° C. for 24hours to about 7 days (168 hours). A 100 μL portion of the reactionmixture was diluted with 1 volume of MeOH and analyzed by HPLC. Theresults of Experiments 1-12 using Method H are summarized in Table 13.

TABLE 6 Method A PLP Amine PRM-102 Time % (I-a) Buffer pH Co-solvent(mM) Enzyme donor (mg) (hrs) 2 Conversion 1 6.5 mg 100 mM 7 MeOH 1ATA-117 D-alanine 39 72 (S) 3% (R¹ = H, (0.85 mL) (0.15 mL) (3 mg) (4.5mg) citrate salt) 2 6.5 mg 100 mM 7 CF₃CH₂OH 1 ATA-117 D-alanine 39 72 —n/a (R¹ = H, (0.85 mL) (0.15 mL) (3 mg) (4.5 mg) citrate salt) 3 20 mg100 mM 7 none 1 ATA-117 D-alanine 60 72 (S) 3% (R¹ = H, (2 mL) (3 mg)(9.5 mg) citrate salt) 4 20 mg 100 mM 7 MeOH 1 ATA-117 D-alanine 60 72(S) 2% (R¹ = H, (1.8 mL) (0.2 mL) (10 mg) (11 mg) citrate salt) 5 20 mg100 mM 7 MeOH 1 broad-range D-alanine 60 72 — n/a (R¹ = H, (1.8 mL) (0.2mL) transaminase (11 mg) citrate salt) (1.7 mg) 6 20 mg 100 mM 7 MeOH 1glutamate D-alanine 60 72 — n/a (R¹ = H, (1.8 mL) (0.2 mL) pyruvate (11mg) citrate salt) transaminase (1 mg) 7 20 mg 100 mM 7 none 1 ATA-113L-alanine 60 72 (S) 15%  (R¹ = H, (2 mL) (10 mg) (9.5 mg) citrate salt)8 20 mg 100 mM 7 MeOH 1 ATA-113 L-alanine 60 72 (S) 5% (R¹ = H, (1.8 mL)(0.2 mL) (10 mg) (11 mg) citrate salt)

TABLE 7 Method B PLP NH₄CO₂H FDH LADH NAD (I-a) Buffer pH co-solvent(mM) enzyme donor (mg) (μL) (μL) (mM) 2 % 1 3.5 mg 100 mM 7 DMSO 1ATA-117 D-alanine 4 25 10 1 — n/a (R¹ = H, (0.85 mL) (0.15 mL) 1 (3 mg)(9.5 mg) citrate salt) 2 3.5 mg 100 mM 7 MeOH 1 ATA-117 D-alanine 4 2510 1 (S) 1% (R¹ = H, (0.85 mL) (0.15 mL) (3 mg) (9.5 mg) citrate salt) 33.5 mg 20 mM 7.5 DMSO 1 ATA-117 D-alanine 4 25 10 1 — n/a (R¹ = H, (0.85mL) (0.15 mL) (3 mg) (9.5 mg) citrate salt) 4 3.5 mg 20 mM 7.5 MeOH 1ATA-117 D-alanine 4 25 10 1 — n/a (R¹ = H, (0.85 mL) (0.15 mL) (3 mg)(9.5 mg) citrate salt) 5 6.5 mg 100 mM 7 CF₃CH₂OH 1 ATA-117 D-alanine 650 20 1 — n/a (R¹ = H, (0.85 mL) (0.15 mL) (3 mg) (13 mg) citrate salt)6 6.5 mg 100 mM 7 MeOH 1 ATA-117 D-alanine 9 50 20 1 (S) 5% (R¹ = H,(0.85 mL) (0.15 mL) (3 mg) (22 mg) citrate salt) 7 3.5 mg 100 mM 7 MeOH1 ATA-113 D-alanine 4 22 8.8 1 (S) 8% (R¹ = H, (0.85 mL) (0.15 mL) (1.3mg) (9.5 mg) citrate salt) 8 3.5 mg 100 mM 7 DMSO 1 ATA-113 D-alanine 422 8.8 1 (S) 37% (R¹ = H, (0.85 mL) (0.15 mL) (1.3 mg) (9.5 mg) citratesalt) 9 10 mg 100 mM 7 DMSO 1 ATA-113 D-alanine 40 216 86 1 (S) 12% (R¹= H, (8.5 mL) (1.5 mL) (13 mg) (95 mg) citrate salt) 10 3.5 mg 100 mM 7DMSO 1 ATA-113 D-alanine 5 22 8.8 1 (S) 10% (R¹ = H, (0.85 mL) (1.3 mL);(1.3 mg) (12 mg) citrate salt) Et₂O (0.2 mL)

TABLE 8 Method C PRM- PLP Amine 102 Time % (I-a) Buffer pH Co-solvent(mM) Enzyme donor (mg) (hrs) 2 Conversion 1 15 mg 100 mM 7 MeOH 1ATA-117 D-alanine 60 24 (S)  2% (R¹ = H) (1.8 mL) (0.2 mL) (10 mg) (13mg) 2 15 mg 100 mM 7 MeOH 1 ATA-113 L-alanine 60 24 (S) 10% (R¹ = H)(1.8 mL) (0.2 mL) (10 mg) (13 mg) 3 1 mg 20 mM 7.5 MeOH 0.5 ATA-113(S)-methyl benzyl none 72 (S)  9% (R¹ = H) (1.2 mL) (0.12 mL) (2 mg)amine (1.4 μL) 4 1 mg 20 mM 7.5 MeOH 0.5 ATA-113 (S)-methyl benzyl 30 72(S) 41% (R¹ = H) (1.2 mL) (0.12 mL) (2 mg) amine (1.4 μL) 5 8.5 mg 20 mM7.5 MeOH 1 ATA-113 (S)-methyl benzyl 300 72 (S) 42% (R¹ = H) (10 mL) (1mL) (20 mg) amine (13 μL) 6 1 mg 20 mM 7.5 MeOH 0.5 ATA-117 (S)-methylbenzyl none 72 — n/a (R¹ = H) (1.2 mL) (0.12 mL) (2 mg) amine (1.4 μL) 71 mg 20 mM 7.5 MeOH 0.5 ATA-117 (S)-methyl benzyl 30 72 — n/a (R¹ = H)(1.2 mL) (0.12 mL) (2 mg) amine (1.4 μL) 8 1 mg 20 mM 7.5 MeOH 0.5ATA-117 (S)-methyl benzyl 60 72 — n/a (R¹ = H) (1.2 mL) (0.12 mL) (4 mg)amine (1.4 μL) 9 8.5 mg 20 mM 7.5 MeOH 0.5 ATA-117 (S)-methyl benzyl 15072 — n/a (R¹ = H) (10 mL) (1 mL) (10 mg) amine (13 μL) 10 8.5 mg 20 mM 8MeOH 0.5 ATA-117 (S)-methyl benzyl 300 72 — n/a (R¹ = H) (9 mL) (1 mL)(20 mg) amine (13 μL) 11 6.4 mg 20 mM 8 MeOH 0.5 ATA-117 (S)-methylbenzyl 300 72 — n/a (R¹ = H) (9 mL) (1 mL) (20 mg) amine (13 μL) 12 4.3mg 20 mM 8 MeOH 0.5 ATA-117 (S)-methyl benzyl 300 72 — n/a (R¹ = H) (9mL) (1 mL) (20 mg) amine (13 μL) 13 2.1 mg 20 mM 8 MeOH 0.5 ATA-117(S)-methyl benzyl 300 72 — n/a (R¹ = H) (9 mL) (1 mL) (20 mg) amine (13μL) 14 1.6 mg 20 mM 7.5 none 1 Vibrio (S)-methyl benzyl none 72 (R)  2%(R¹ = H) (1.2 mL) fluvialis (55 μL) amine (13 μL) 15 1 mg 20 mM 7.5 MeOH0.5 Vibrio (R)-methyl benzyl none 72 (R) 10% (R¹ = H) (1.2 mL) (0.12 mL)Fluvialis amine (100 μL) (1.4 μL) 16 4.3 mg 20 mM 7.5 MeOH 0.5 Vibrio(R)-methyl benzyl 140 72 (R) 15% (R¹ = H) (4.95 mL) (0.55 mL) Fluvialisamine (155 μL) (6.5 μL) 17 1.6 mg 20 mM 7.5 none 0.5 Vibrio (S)-methylbenzyl none 72 (R)  3% (R¹ = H) (2 mL) Fluvialis amine (100 μL) (1.4 μL)18 8.5 mg 20 mM 7.5 MeOH 0.5 Vibrio (R)-methyl benzyl none 72 (R) 10%(R¹ = H) (10 mL) (1 mL) Fluvialis amine (100 μL) (13 μL) 19 8.5 mg 20 mM7.5 MeOH 0.5 Vibrio (S)-methyl benzyl none 72 (R) 10% (R¹ = H) (10 mL)(1 mL) Fluvialis amine (310 μL) (13 μL) 20 8.5 mg 20 mM 7.5 MeOH 0.5Vibrio (S)-methyl benzyl 275 72 (R) 10% (R¹ = H) (10 mL) (1 mL)Fluvialis amine (310 μL) (13 μL) 21 14 mg 20 mM 7.5 MeOH 0.5 Vibrio(R)-methyl benzyl 300 72 (R) 17% (R¹ = H, (10 mL) (1 mL) Fluvialis aminecitrate salt) (310 μL) (13 μL) 22 7 mg 20 mM 7.5 MeOH 0.5 Vibrio(R)-methyl benzyl 150 144 (R) 28% (R¹ = H, (5 mL) (0.5 mL) Fluvialisamine citrate salt) (155 μL) (7 μL) 23 5.5 mg 20 mM 7.5 EtOAc 1 Vibrio(R)-methyl benzyl 150 72 — n/a (R¹ = Bn) (5 mL) (0.5 mL) Fluvialis amine(150 μL) (7 μL) 24 5.5 mg 20 mM 7.5 cyclohexanone 1 Vibrio (R)-methylbenzyl 150 72 — n/a (R¹ = Bn) (5 mL) (0.5 mL) Fluvialis amine (150 μL)(7 μL) 25 8.5 mg 20 mM 7 MeOH 0.5 ATA-113 (S)-methyl benzyl 300 72 (S)1.7%  (R¹ = Bn) (10 mL) (1 mL) (20 mg) amine (13 μL) 26 8.5 mg 20 mM 7MeOH 0.5 ATA-113 (S)-methyl benzyl 300 72 (S)  2% (R¹ = CBz) (10 mL) (1mL) (20 mg) amine (13 μL) 27 8.5 mg 20 mM 7 MeOH 0.5 ATA-117 (S)-methylbenzyl 300 72 — n/a (R¹ = Bn) (10 mL) (1 mL) (20 mg) amine (13 μL) 288.5 mg 20 mM 7 MeOH 0.5 ATA-117 (S)-methyl benzyl 300 72 — n/a (R¹ =Cbz) (10 mL) (1 mL) (20 mg) amine (13 μL)

TABLE 9 Method D PRM- Co- PLP Amine 102 Time % (I-a) Buffer pH solvent(mM) Enzyme Donor (5 equiv.) (mg) (hrs) 2 Conversion 1 14 mg 20 mM 7.0MeOH 0.5 Vibrio L-alanine 300 mg 168 (R) 16.1%  (R¹ = H, (9 mL) (1.0 mL)Fluvialis citrate salt) (310 μL) 2 14 mg 20 mM 7.0 MeOH 0.5 VibrioD-alanine 300 mg 168 (R) 12.7%  (R¹ = H, (9 mL) (1.0 mL) Fluvialiscitrate salt) (310 μL) 3 14 mg 20 mM 7.0 MeOH 0.5 VibrioR-Methylbenzylamine 300 mg 168 (R) 14.1%  (R¹ = H, (9 mL) (1.0 mL)Fluvialis citrate salt) (310 μL) 4 14 mg 20 mM 7.0 MeOH 0.5 VibrioS-Methyl benzylamine 300 mg 168 (R) 7.6% (R¹ = H, (9 mL) (1.0 mL)Fluvialis citrate salt) (310 μL) 5 14 mg 20 mM 7.0 MeOH 0.5 Vibrio2-aminoindane 300 mg 168 (R) 4.1% (R¹ = H, (9 mL) (1.0 mL) Fluvialiscitrate salt) (310 μL) 6 14 mg 20 mM 7.0 MeOH 0.5 Vibrio S-1-Aminoindane300 mg 168 — n/a (R¹ = H, (9 mL) (1.0 mL) Fluvialis citrate salt) (310μL) 7 14 mg 20 mM 7.0 MeOH 0.5 Vibrio R-1-Aminoindane 300 mg 168 (R)5.1% (R¹ = H, (9 mL) (1.0 mL) Fluvialis citrate salt) (310 μL) 8 14 mg20 mM 7.0 MeOH 0.5 Vibrio R-2-Amino-1-propanol 300 mg 168 (R) 3.8% (R¹ =H, (9 mL) (1.0 mL) Fluvialis citrate salt) (310 μL) 9 14 mg 20 mM 7.0MeOH 0.5 Vibrio S-2-Amino-1-propanol 300 mg 168 (R) 4.6% (R¹ = H, (9 mL)(1.0 mL) Fluvialis citrate salt) (310 μL) 10 14 mg 20 mM 7.0 MeOH 0.5Vibrio (1R,2S)-cis-1-Amino-2- 300 mg 168 — n/a (R¹ = H, (9 mL) (1.0 mL)Fluvialis indanol citrate salt) (310 μL) 11 14 mg 20 mM 7.0 MeOH 0.5Vibrio (1R,2R)-trans-1-Amino- 300 mg 168 — n/a (R¹ = H, (9 mL) (1.0 mL)Fluvialis 2-indanol citrate salt) (310 μL) 12 14 mg 20 mM 7.0 MeOH 0.5Vibrio 1-R-Amino-6- 300 mg 168 — n/a (R¹ = H, (9 mL) (1.0 mL) Fluvialishydroxyindanamine citrate salt) (310 μL) 13 14 mg 20 mM 7.0 MeOH 0.5Vibrio Isopropylamine 300 mg 168 (R) 8.4% (R¹ = H, (9 mL) (1.0 mL)Fluvialis citrate salt) (310 μL)

TABLE 10 Method E PRM- PLP Amine 102 Time % (I-a) Buffer pH Co-solvent(mM) Enzyme donor (mg) (hrs) 2 Conversion 1 14 mg 20 mM 7.0 MeOH 0.5Vibrio L-alanine 300 mg 168 (R) 6.4% (R¹ = H, (9 mL) (1.0 mL) Fluvialis(10 mg) citrate salt) (155 μL) 2 14 mg 20 mM 8.0 MeOH 0.5 VibrioL-alanine 300 mg 168 (R) 14.9% (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10mg) citrate salt) (155 μL) 3 14 mg 20 mM 9.0 MeOH 0.5 Vibrio L-alanine300 mg 168 (R) 10.2% (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10 mg) citratesalt) (155 μL) 4 14 mg 100 mM 8.1 MeOH 0.5 Vibrio L-alanine 300 mg 168(R) 3.6% (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10 mg) citrate salt) (155μL) 5 14 mg 100 mM 7.1 MeOH 0.5 Vibrio L-alanine 300 mg 168 (R) 7.9% (R¹= H, (9 mL) (1.0 mL) Fluvialis (10 mg) citrate salt) (155 μL)

TABLE 11 Method F PRM- PLP Amine 102 Time % (I-a) Buffer pH Co-solvent(mM) Enzyme donor (mg) (hrs) 2 Conversion 1 14 mg 20 mM 7.5 MeOH 0.5Vibrio D-alanine 300 mg 168 (R) 12.7% (R¹ = H, (9 mL) (1.0 mL) Fluvialis(10 mg) citrate salt) (155 μL) 2 14 mg 20 mM 7.5 MeOH none VibrioD-alanine 300 mg 168 — n/a (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10 mg)citrate salt) (155 μL) 3 14 mg 20 mM 7.5 MeOH 0.5 Vibrio L-alanine 300mg 168 (R) 16.1% (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10 mg) citratesalt) (155 μL) 4 14 mg 20 mM 7.0 MeOH none Vibrio L-alanine 300 mg 168 —n/a (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10 mg) citrate salt) (155 μL) 514 mg 20 mM 7.5 MeOH 0.5 Vibrio L-alanine 300 mg 168 (R)   31% (R¹ = H,(9 mL) (1.0 mL) Fluvialis (10 mg) citrate salt) (310 μL) 6 14 mg 20 mM7.5 MeOH 0.5 Vibrio L-alanine 300 mg 168 (R) 41.1% (R¹ = H, (9 mL) (1.0mL) Fluvialis (10 mg) citrate salt) (310 μL on day 1, 155 μL on day 4) 714 mg 20 mM 7.5 MeOH 0.5 Vibrio L-alanine 300 mg 168 (R) 48.7% (R¹ = H,(9 mL) (1.0 mL) Fluvialis, (10 mg) citrate salt) NEW BATCH (310 μL onday 1, 155 μL on day 6) 8 45 mg 20 mM 7.5 MeOH 0.5 Vibrio L-alanine 300mg 168 (R)  8.6% (R¹ = H, (9 mL) (1.0 mL) Fluvialis (10 mg) citratesalt) NEW BATCH (310 μL) 9 14 mg 20 mM 7.5 MeOH 0.5 Vibrio L-alanine 300mg 168 (R) 14.5% (R¹ = H, (9 mL) (1.0 mL) Fluvialis, (20 mg) citratesalt) NEW BATCH (310 μL)

TABLE 12 Method G PRM- PLP Amine 102 Time % (I-a) Buffer AdditivesCo-solvent (mM) Enzyme donor (mg) (hrs) 2 Conversion 1 14 mg 20 mMAmberlite MeOH 0.5 Vibrio L-alanine 300 mg 96 (R) 3.7% (R¹ = H, (9 mL)Resin (100 wt (1.0 mL) Fluvialis (10 mg) citrate salt) %) (310 μL) 2 14mg 20 mM Dowex Resin MeOH 0.5 Vibrio L-alanine 300 mg 96 — n/a (R¹ = H,(9 mL) (100 wt %) (1.0 mL) Fluvialis (10 mg) citrate salt) (310 μL) 3 14mg 20 mM β- MeOH 0.5 Vibrio L-alanine 300 mg 96 (R) 14.0 (R¹ = H, (9 mL)Cyclodextrin (1.0 mL) Fluvialis (10 mg) citrate salt) (100 wt %) (310μL) 4 14 mg 20 mM γ- MeOH 0.5 Vibrio L-alanine 300 mg 96 (R) 11.4 (R¹ =H, (9 mL) Cyclodextrin (1.0 mL) Fluvialis (10 mg) citrate salt) (100 wt%) (310 μL) 5 14 mg 20 mM Sodium MeOH 0.5 Vibrio benzylamine 300 mg 96 —n/a (R¹ = H, (9 mL) Bisulfate (100 wt (1.0 mL) Fluvialis (10 mg) citratesalt) %) (310 μL)

TABLE 13 Method H PRM- PLP Amine 102 Time % (I-a) Buffer pH Co-solvent(mM) Enzyme donor (mg) (hrs) 2 Conversion 1 14 mg 20 mM 7.5 none 0.5Vibrio L-alanine 300 mg 168 (R) 6.4% (R¹ = H, (9 mL) Fluvialis (10 mg)citrate salt) (155 μL) 2 14 mg 20 mM 7.5 MeOH 0.5 Vibrio L-alanine 300mg 168 (R) 14.9%  (R¹ = H) (9 mL) (1.0 mL) Fluvialis (10 mg) (155 μL) 314 mg 20 mM 7.5 DMSO 0.5 Vibrio L-alanine 300 mg 168 (R) 8.2% (R¹ = H)(9 mL) (1.0 mL) Fluvialis (10 mg) (155 μL) 4 14 mg 100 mM 7.5 Heptane0.5 Vibrio L-alanine 300 mg 168 — n/a (R¹ = H) (9 mL) (2.0 mL) Fluvialis(10 mg) (155 μL) 5 14 mg 100 mM 7.5 Heptane 0.5 Vibrio R-Methyl 300 mg168 — n/a (R¹ = H) (9 mL) (2.0 mL) Fluvialis benzylamine (155 μL) 6 14mg 100 mM 7.5 Toluene 0.5 Vibrio L-alanine 300 mg 168 — n/a (R¹ = H) (9mL) (2.0 mL) Fluvialis (10 mg) (155 μL) 7 14 mg 100 mM 7.5 Toluene 0.5Vibrio R-Methyl 300 mg 168 — n/a (R¹ = H) (9 mL) (2.0 mL) Fluvialisbenzylamine (155 μL) 8 14 mg 100 mM 7.5 2-Methyl 0.5 Vibrio L-alanine300 mg 168 — n/a (R¹ = H) (9 mL) tetrahydrofuran Fluvialis (10 mg) (2.0mL) (155 μL) 9 14 mg 100 mM 7.5 2-Methyl 0.5 Vibrio R-Methyl 300 mg 168— n/a (R¹ = H) (9 mL) tetrahydrofuran Fluvialis benzylamine (2.0 mL)(155 μL) 10 14 mg 100 mM 7.5 Ethylacetate 0.5 Vibrio L-alanine 300 mg168 (R) 12.9% (aq. layer) (R¹ = H) (9 mL) (2.0 mL) Fluvialis (10 mg)1.1% (organic (155 μL) layer) 11 14 mg 100 mM 7.5 Hexanes 0.5 VibrioL-alanine 300 mg 168 (R) 4.2% (aq. layer) (R¹ = H) (9 mL) (2.0 mL)Fluvialis (10 mg) 1.6% (organic (155 μL) layer) 12 14 mg 100 mM 7.5Perfluorohexane 0.5 Vibrio L-alanine 300 mg 168 — n/a (R¹ = H) (9 mL)(2.0 mL) Fluvialis (10 mg) (155 μL) Table Legend PLP pyridoxal phosphatePRM 102 pyruvate reductase mix available from Codexis, Inc. (R) (R)isomer preferentially generated (S) (S) isomer preferentially generated% percent conversion of the preferred isomer as determined by HPLC n/ano product detected by HPLC Me methyl, —CH₃ Bn benzyl, —CH₂C₆H₅ Cbzcarbobenzyloxy, —C(═O)OCH₂C₆H₅ DMSO dimethylsulfoxide MeOH methanolEtOAc ethyl acetate

Example: Evaluation of Candidate Transaminases.

Candidate transaminases were evaluated using the reaction conditionsdescribed in Method A above (see Table 6). These experiments werecarried out using PRM-102 (powder form) as the co-enzyme system andD-alanine or L-alanine as the amino donor molecule. In all but two ofthe runs, a water miscible co-solvent (methanol or trifluoroethanol(CF₃CH₂OH)) was employed. As shown in Table 6, % conversions of 5% and15% were observed when the tranamination was conducted in the presenceof omega amine transaminase enzyme ATA-113, and % conversions of 2% and3% were observed when the tranamination was conducted in the presence ofomega amine transaminase enzyme ATA-117. Omega amine transaminaseenzymes ATA-117, known generally to be an (R)-selective transaminase,produced the compound (S)-(II-a) rather than compound (R)-(II-a).ATA-113, known generally to be an (S)-selective transaminase, producedthe compound (S)-(II-a). The descriptor “n/a” indicates that thetransamination product (II-a) was not detected by HPLC in the reactionmixture.

Example: Evaluation of FDH/LADH/NAD Coenzyme Mixtures.

Candidate mixtures of the co-enzymes FDH, LADH and NAD were evaluatedusing the reaction conditions described in Method B above (see Table 7).These experiments were carried out using ATA-117 and ATA-113 as theamine transferase enzyme; D-alanine as the amino donor molecule; andDMSO, methanol or trifluoroethanol as the co-solvent. As shown in Table7, % conversions of from 1% to 37% were observed using this co-enzymesystem. Further, transamination was achieved using either ATA-117 orATA-113 as the transaminase enzyme. The descriptor “n/a” indicates thatthe transamination product (II-a) was not detected by HPLC in thereaction mixture.

Example: Comparison of Omega Transaminase from Vibrio fluvialis withATA-117 and ATA-113.

The activity of the omega transaminase from Vibrio fluvialis wascompared with the activities of ATA-117 and ATA-113 using the reactionconditions described in Method C above (see Table 8). These experimentswere carried out using PRM-102 as the co-enzyme system and one of thefollowing amino donor molecule: D-alanine, L-alanine, (5)-methylbenzylamine and (R)-methyl benzylamine. When ATA-117 or ATA-113 wasemployed as the transaminase enzyme, compound (S)-(II-a) was produced.As indicated in Table 8, this product stereochemistry (S) was obtainedwith amino donor molecules D-alanine, L-alanine, and (S)-methylbenzylamine. In contrast, when the omega amine transaminase from Vibriofluvialis was employed as the amine transaminase enzyme, compound(R)-(II-a) was produced. As indicated in Table 8, this productstereochemistry was obtained when either (R) or (S)-methyl benzylaminewas used as the amino donor molecule. The descriptor “n/a” indicatesthat the transamination product (II-a) was not detected by HPLC in thereaction mixture.

Example: Evaluation of Candidate Amino Donor Molecules.

Candidate amino donor molecules were evaluated using the reactionconditions described in Method D above (see Table 9). These experimentswere carried out using the omega transaminase from Vibrio fluvialis; a20 mM phosphate buffer (pH=7.0); and methanol as the co-solvent.L-alanine, D-alanine, (R)-methylbenzylamine, (S)-methylbenzylamine,2-aminoindane, (R)-1-aminoindane, (R)-2-amino-1-propanol,(S)-2-amino-1-propanol, (1R,2S)-cis-1-amino-2-indanol,(1R,2R)-trans-1-amino-2-indanol, 1-(R)-amino-6-hydroxyindanamine andisopropylamine were tested. As indicated in Table 9, only compound(R)-(II-a) was observed. L-alanine as amino donor molecule provided a %conversion of 16.1% over a period of 7 days. The descriptor “n/a”indicates that the transamination product (II-a) was not detected byHPLC in the reaction mixture.

Example: Evaluation of Candidate Reaction pH and Buffer Strengths.

Candidate reaction pH and buffer strengths were evaluated using thereaction conditions described in Method E above (see Table 10). Theseexperiments were carried out using buffers with different pH's andmolarities. As shown in Table 10, a 20 mM phosphate buffer (maintaininga pH=8.0) was found to provide a % conversion of 14.9%. Increasing themolarity of the buffer (100 mM) was found to cause precipitation of thestarting material (I-a) and reduce conversion to the product (R)-(II-a).The descriptor “n/a” indicates that the transamination product (II-a)was not detected by HPLC in the reaction mixture.

Table 11 shows the results for transamination reactions conducted atpH=7.5. These reactions were monitored for any change in the pH, and anychange in the pH of the reaction mixture was adjusted using 0.1 N sodiumhydroxide solution (Method F). However, it was also found that theconversion was dependent on the age of the enzyme. A fresh bottle ofenzyme was found to increase % conversion up to 31% using L-alanine overa period of 7 days and 2 units/mg of the substrate compound (I-a). Whenthe reaction mixture was charged with an additional unit of fresh enzymeat the end of day 6, the conversion improved to 48.7%. The descriptor“n/a” indicates that the transamination product (II-a) was not detectedby HPLC in the reaction mixture.

Example: Evaluation of Candidate Additives.

Candidate additives were evaluated using the reaction conditionsdescribed in Method G above (see Table 12). These experiments werecarried out using additives, such as resins, cyclodextrins and sulfites.Resins capable of adsorbing the starting material compound (I-a) as wellas the product compound (H-a) were screened. In addition, β- andγ-cyclodextrins that are known to solubilize organic compounds were alsotested (% conversions of 14.0% and 11.4% were observed for β- andγ-cyclodextrin, respectively). The descriptor “n/a” indicates that thetransamination product (II-a) was not detected by HPLC in the reactionmixture.

Example: Evaluation of Candidate Co-Colvents.

Candidate co-colvents were evaluated using the reaction conditionsdescribed in Method H above (see Table 13). For monophasic systems, itwas found that using methanol as the co-solvent provided the highest %conversion of those tested. For biphasic systems, it was found thatusing ethyl acetate as the co-solvent provided the highest % conversionof those tested. The descriptor “n/a” indicates that the transaminationproduct (II-a) was not detected by HPLC in the reaction mixture.

Exemplary Sulfonylation of a Compound of Formula (II)

Procedure described in Tremblay et al., “Discovery of a Potent andOrally Active Hedgehog Pathway Antagonist (IPI-926)” J. Med. Chem.(2009) 52:4400-4418, incorporated herein by reference.

A solution of amine (R¹=CBz) (5.10 g, 9.09 mmol, 1 equiv) indichloromethane (60 mL) was treated with diisopropylethylamine (5.88 g,45.5 mmol, 5.0 equiv), cooled to 0° C., and treated with methanesulfonylchloride (2.08 g, 18.2 mmol, 2.0 equiv). The reaction mixture wasstirred for 30 min and partitioned between saturated aqueous sodiumbicarbonate and ethyl acetate. The organic layer was separated, driedover sodium sulfate, and concentrated to dryness to provide a cruderesidue. The residue was purified using silica gel chromatography(10-30% EtOAc/hexanes) to provide the N-Cbz sulfonylated product. Asuspension of the isolated product and 10% palladium on carbon (1.0 g)in 2-propanol (50 mL) was placed under hydrogen atmosphere and stirredfor 4 h at room temperature. The reaction mixture was then filtered onCelite and the filtrate concentrated to dryness. The residue was thenpurified using silica gel chromatography (0-5% DCM/MeOH) to giveIPI-926-(4.06 g, 8.05 mmol, 95% for two steps). NMR δH (400 MHz, CDCl₃)6.90 (br s, 1H), 3.31 (dt, J=10.6, 3.8 Hz, 1H), 3.20 (br s, 1H), 3.10(dd, J=13.7, 4.5 Hz, 1H), 2.91 (s, 3H), 2.62 (dd, J=9.9, 7.6 Hz, 1H),2.33 (br d, J=14.5 Hz, 1H), 2.27-2.15 (m, 1H), 2.10 (dd, J=14.5, 6.9 Hz,1H), 1.99-1.17 (m, 28H), 1.05 (q, J=11.6 Hz, 1H), 0.93 (d, J=7.4 Hz,3H), 0.88 (d, J=6.6 Hz, 3H), 0.86 (s, 3H); NMR δC (100 MHz, CDCl₃)140.47, 124.53, 82.48, 76.97, 63.73, 54.08, 53.87, 50.12, 49.98, 47.19,44.73, 42.27, 42.10, 40.24, 37.55, 37.44, 36.04, 34.44, 31.87, 31.33,30.46, 29.79, 28.37, 27.94, 26.26, 24.19, 22.70, 18.92, 10.19;m/z=505.29 [M+H]⁺; HPLC 99.1 a/a % at 215 nm.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

1.-38. (canceled)
 39. A compound of formula (II):

or a salt thereof made by a process comprising contacting a compound of formula (I):

or a salt thereof, an amino donor molecule, and an amine transaminase enzyme in a solution to provide a compound of formula (II) or a salt thereof; wherein: R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR¹⁶, —C(O)R¹⁶, —CO₂R¹⁶, —SO₂R¹⁶, —C(O)N(R¹⁷)(R¹⁷), —[C(R¹⁶)₂]_(q)—R¹⁶, —[(W)—N(R¹⁷)C(O)]_(q)R¹⁶, —[(W)—C(O)]_(q)R¹⁶, —[(W)—C(O)]_(q)R¹⁶, —[(W)—C(O)]_(q)R¹⁶, —[(W)—SO₂]_(q)R¹⁶, —[(W)—N(R¹⁷)SO₂]_(q)R¹⁶, —[(W)—C(O)N(R¹⁷)]_(q)R¹⁷, —[(W)—O]_(q)R¹⁶, —[(W)—N(R¹⁷)]_(q)R¹⁶, or —[(W)—S]_(q)R¹⁶; wherein W is a diradical and q is 1, 2, 3, 4, 5, or 6; each R² and R³ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, halo, —OR¹⁶, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶, or R² and R³ taken together form a double bond or form a group

wherein Z is NR¹⁷, O, or C(R¹⁸)₂; R⁴ is independently H, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; each R⁵ and R⁶, is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; or R⁵ and R⁶ taken together with the carbon to which they are bonded form C═O, C═S, C═N—OR¹⁷, C═N—R¹⁷, C═N—N(R¹⁷)₂, or form an optionally substituted 3-8 membered ring; each R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; or R¹⁰ and R¹¹ taken together, or R¹¹ and R¹² taken together, form a double bond or form a group

wherein Z is NR¹⁷, O, or C(R¹⁵)₂; each R¹⁴ and R¹⁵ is, independently, H, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; or R¹⁴ and R¹⁵ taken together with the carbon to which they are bonded form C═O or C═S; X is a bond or the group —C(R¹⁹)₂—; wherein each R¹⁹ is, independently, H, alkyl, aralkyl, halo, —CN, —OR¹⁶, or —N(R¹⁷)₂; R¹⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or —[C(R²⁰)₂]_(p)—R²¹ wherein p is 0-6; or any two occurrences of R¹⁶ on the same substituent are taken together to form a 4-8 membered optionally substituted ring; R¹⁷ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁰, C(═O)OR²³, —SO₂R²⁰, —C(═O)N(R²⁰)₂, or —[C(R²⁰)₂]_(p)—R²¹ wherein p is 0-6; or any two occurrences of R¹⁷ on the same substituent are taken together to form a 4-8 membered optionally substituted ring; R¹⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —CN, —OR²⁰, —OSi(R²⁰)₃, —C(═O)R²⁰, —C(═O)OR²⁰, —SO₂R²⁰ or —C(═O)N(R²⁰)₂; R²⁰ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any two occurrences of R²⁰ on the same substituent are taken together to form a 4-8 membered optionally substituted ring; R²¹ is —OR²², —N(R²²)C(═O)R²², —N(R²²)C(═O)OR²², —N(R²²)SO₂(R²²), —C(═O)R²²N(R²²)₂, —OC(═O)R²²N(R²²)(R²²), —SO₂N(R²²)(R²²), —N(R²²)(R²²), —C(═O)OR²², —C(═O)N(OH)(R²²), —OS(O)₂OR²², —S(O)₂OR²², —OP(═O)(OR²²)(OR²²), —N(R²²)P(O)(OR²²)(OR²²), or —P(═O)(OR²²)(OR²²); and R²² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl; or any two occurrences of R²² on the same substituent are taken together to form a 4-8 membered optionally substituted ring.
 40. A compound of formula (III):

or salt thereof made by a process comprising contacting a compound of formula (I):

or a salt thereof, an amino donor molecule, and an amine transaminase enzyme in a solution to provide a compound of formula (II):

or a salt thereof; further comprising contacting a compound of formula (II) or a salt thereof with a sulfonylating agent that is methanesulfonyl chloride or methanesulfonyl anhydride to provide a compound of formula (III) or salt thereof; wherein: R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR¹⁶, —C(O)R¹⁶, —CO₂R¹⁶, —SO₂R¹⁶, —C(O)N(R¹⁷)(R¹⁷), —[C(R¹⁶)₂]_(q)—R¹⁶, —[(W)—N(R¹⁷)C(O)]_(q)R¹⁶, —[(W)—C(O)]_(q)R¹⁶, —[(W)—C(O)O]_(q)R¹⁶, —[(W)—OC(O)]_(q)R¹⁶, —[(W)—SO₂]_(g)R¹⁶, —[(W)—N(R¹⁷)SO₂]_(q)R¹⁶, —[(W)—C(O)N(R¹⁷)]_(q)R¹⁷, —[(W)—O]_(q)R¹⁶, —[(W)—N(R¹⁷)]_(q)R¹⁶, or —[(W)S]_(q)R¹⁶; wherein W is a diradical and q is 1, 2, 3, 4, 5, or 6; each R² and R³ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, halo, —OR¹⁶, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶, or R² and R³ taken together form a double bond or form a group

wherein Z is NR¹⁷, O, or C(R¹⁸)₂; R⁴ is independently H, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; each R⁵ and R⁶, is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; or R⁵ and R⁶ taken together with the carbon to which they are bonded form C═O, C═S, C═N—OR¹⁷, C═N—R¹⁷, C═N—N(R¹⁷)₂, or form an optionally substituted 3-8 membered ring; each R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; or R¹⁰ and R¹¹ taken together, or R¹¹ and R¹² taken together, form a double bond or form a group

wherein Z is NR¹⁷, O, or C(R¹⁸)₂; each R¹⁴ and R¹⁵ is, independently, H, halo, —OR¹⁶, —N(R¹⁷)₂, or —SR¹⁶; or R¹⁴ and R¹⁵ taken together with the carbon to which they are bonded form C═O or C═S; X is a bond or the group —C(R¹⁹)₂—; wherein each R¹⁹ is, independently, H, alkyl, aralkyl, halo, —CN, —OR¹⁶, or —N(R¹⁷)₂; R¹⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or —[C(R²⁰)₂]_(p)—R²¹ wherein p is 0-6; or any two occurrences of R¹⁶ on the same substituent are taken together to form a 4-8 membered optionally substituted ring; R¹⁷ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R²⁰, —C(═O)OR²⁰, —SO₂R²⁰, —C(═O)N(R²⁰)₂, or —[C(R²⁰)₂]_(p)—R²¹ wherein p is 0-6; or any two occurrences of R¹⁷ on the same substituent are taken together to form a 4-8 membered optionally substituted ring; R¹⁸ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —CN, —OR²⁰, —OSi(R²⁰)₃, —C(═O)R²⁰, —C(═O)OR²⁰, —SO₂R²⁰ or —C(═O)N(R²⁰)₂; R²⁰ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any two occurrences of R²⁰ on the same substituent are taken together to form a 4-8 membered optionally substituted ring; R²¹ is —OR²², —N(R²²)C(═O)R²², —N(R²²)C(═O)OR²², —N(R²²)SO₂(R²²), —C(═O)R²²N(R²²)₂, —OC(═O)R²²N(R²²)(R²²), —SO₂N(R²²)(R²²), —N(R²²)(R²²), —C(═O)OR²², —C(═O)N(OH)(R²²), —OS(O)₂OR²², —S(O)₂OR²², —OP(═O)(OR²²)(OR²²), —N(R²²)P(O)(OR²²)(OR²²), or —P(═O)(OR²²)(OR²²); R²² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl; or any two occurrences of R²² on the same substituent are taken together to form a 4-8 membered optionally substituted ring; and R²³ is —CH₃. 